Method for optically controlling a neuron with a mammalian codon optimized nucleotide sequence that encodes a variant opsin polypeptide derived from natromonas pharaonis (NpHR)

ABSTRACT

Stimulation of target cells using light, e.g., in vivo or in vitro, is implemented using a variety of methods and devices. One example involves a vector for delivering a light-activated NpHR-based molecule comprising a nucleic acid sequence that codes for light-activated NpHR-based molecule and a promoter. Either a high expression of the molecule manifests a toxicity level that is less than about 75%, or the light-activated NpHR-based proteins are expressed using at least two NpHR-based molecular variants. Each of the variants characterized in being useful for expressing a light-activated NpHR-based molecule that responds to light by producing an inhibitory current to dissuade depolarization of the neuron. Other aspects and embodiments are directed to systems, methods, kits, compositions of matter and molecules for ion pumps or for controlling inhibitory currents in a cell (e.g., in in vivo and in vitro environments).

RELATED PATENT DOCUMENTS

This application is a continuation of U.S. patent application Ser. No.13/855,413, now U.S. Pat. No. 9,284,353, which is a continuation of U.S.patent application Ser. No. 13/208,419, filed Aug. 12, 2011, which is acontinuation of U.S. patent application Ser. No. 12/041,628, filed Mar.3, 2008, and claims benefit under 35 U.S. §119(e) of U.S. ProvisionalApplication Ser. No. 60/904,303 filed on Mar. 1, 2007, whichapplications are incorporated by reference herein in their entirety.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contract OD000616awarded by the National Institutes of Health. The Government has certainrights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing and identified as follows: One27,705 Byte ASCII (Text) file named “stfd-165PA_ST25” created on Nov.19, 2010.

FIELD OF THE INVENTION

The present invention relates generally to systems and approaches forstimulating target cells, and more particularly to using optics tostimulate the target cells.

BACKGROUND

The stimulation of various cells of the body has been used to produce anumber of beneficial effects. One method of stimulation involves the useof electrodes to introduce an externally generated signal into cells.One problem faced by electrode-based brain stimulation techniques is thedistributed nature of neurons responsible for a given mental process.Conversely, different types of neurons reside close to one another suchthat only certain cells in a given region of the brain are activatedwhile performing a specific task. Alternatively stated, not only doheterogeneous nerve tracts move in parallel through tight spatialconfines, but the cell bodies themselves may exist in mixed, sparselyembedded configurations. This distributed manner of processing seems todefy the best attempts to understand canonical order within the centralnervous system (CNS), and makes neuromodulation a difficult therapeuticendeavor. This architecture of the brain poses a problem forelectrode-based stimulation because electrodes are relativelyindiscriminate with regards to the underlying physiology of the neuronsthat they stimulate. Instead, physical proximity of the electrode polesto the neuron is often the single largest determining factor as to whichneurons will be stimulated. Accordingly, it is generally not feasible toabsolutely restrict stimulation to a single class of neuron lasingelectrodes.

Another issue with the use of electrodes for stimulation is that becauseelectrode placement dictates which neurons will be stimulated,mechanical stability is frequently inadequate, and results in leadmigration of the electrodes from the targeted area. Moreover, after aperiod of time within the body, electrode leads frequently becomeencapsulated with glial cells, raising the effective electricalresistance of the electrodes, and hence the electrical power deliveryrequired to reach targeted cells. Compensatory increases in voltage,frequency or pulse width, however, may spread the electrical current andincrease the unintended stimulation of additional cells.

Another method of stimulus uses photosensitive bio-molecular structuresto stimulate target cells in response to light. For instance, lightactivated proteins can be used to control the flow of ions through cellmembranes. By facilitating or inhibiting the flow of positive ornegative ions through cell membranes, the cell can be brieflydepolarized, depolarized and maintained in that state, orhyperpolarized. Neurons are an example of a type of cell that uses theelectrical currents created by depolarization to generate communicationsignals (i.e., nerve impulses). Other electrically excitable cellsinclude skeletal muscle, cardiac muscle, and endocrine cells. Neuronsuse rapid depolarization to transmit signals throughout the body and forvarious purposes, such as motor control (e.g., muscle contractions),sensory responses (e.g., touch, hearing, and other senses) andcomputational functions (e.g., brain functions). Thus, the control ofthe depolarization of cells can be beneficial for a number of differentpurposes, including (but not limited to) psychological therapy, musclecontrol and sensory functions.

SUMMARY

The claimed invention is directed to photosensitive bio-molecularstructures and related methods. The present invention is exemplified ina number of implementations and applications, some of which aresummarized below.

According to one example embodiment of the present invention, animplantable arrangement is implemented having a light-generation devicefor generating light. The arrangement also has a biological portion thatmodifies target cells for stimulation in response to light generated bythe light-generation means in vivo.

According to another example embodiment of the present invention, targetcells are stimulated using an implantable arrangement. The arrangementincludes an electrical light-generation means for generating light and abiological portion. The biological portion has a photosensitivebio-molecular arrangement that responds to the generated light bystimulating target cells in vivo. Stimulation may be manifest as eitherup-regulation, or down-regulation of activity at the target.

According to another example embodiment of the present invention, animplantable device delivers gene transfer vector, such as a virus, whichinduces expression of photosensitive bio-molecular membrane proteins.The device has a light generator, responsive to (for example, charged byor triggered by) an external signal, to generate light and a biologicalarrangement that includes the photosensitive bio-molecular protein thatresponds to the generated light by interacting with target cells invivo. In this manner, the electronic portions of the device may be usedto optically stimulate target cells. Stimulation may be manifested aseither upregulation (e.g., increased neuronal firing activity), ordownregulation (e.g., neuronal hyperpolarization, or alternatively,chronic depolarization) of activity at the target.

According to another example embodiment of the present invention, amethod is implemented for stimulating target cells using photosensitiveproteins that bind with the target cells. The method includes a step ofimplanting the photosensitive proteins and a light generating devicenear the target cells. The light generating device is activated and thephotosensitive protein stimulates the target cells in response to thegenerated light.

Applications include those associated with any population ofelectrically-excitable cells, including neurons, skeletal, cardiac, andsmooth muscle cells, and insulin-secreting pancreatic beta cells. Majordiseases with altered excitation-effector coupling include heartfailure, muscular dystrophies, diabetes, pain, cerebral palsy,paralysis, depression, and schizophrenia. Accordingly, the presentinvention has utility in the treatment of a wide spectrum of medicalconditions, from Parkinson's disease and brain injuries to cardiacdysrhythmias, to diabetes, and muscle spasm.

According to other example embodiments of the present invention, methodsfor generating an inhibitory neuron-current flow involve, in a neuron,engineering a protein that responds to light by producing an inhibitorycurrent to dissuade depolarization of the neuron. In one such method,the protein is halorhodopsin-based and in another method the protein isan inhibitory protein that uses an endogenous cofactor.

According to another example embodiment of the present invention, amethod for controlling action potential of a neuron involves thefollowing steps: engineering a first light responsive protein in theneuron; producing, in response to light, an inhibitory current in theneuron that is generated from the first light responsive protein;engineering a second light responsive protein in the neuron; andproducing, in response to light, an excitation current in the neuronfrom the second light responsive protein.

In another method for controlling a voltage level across a cell membraneof a cell, the method comprises: engineering a first light responsiveprotein in the cell; measuring the voltage level across the cellmembrane; and producing, in response to light of a first wavelength andusing the first light responsive protein, a current across the cellmembrane that is responsive to the measured voltage level.

According to another example embodiment of the present invention, amethod for generating an inhibitory-current flow in neurons isimplemented. The method includes in a neuron, engineering an inhibitoryprotein that responds to light by producing an inhibitory current todissuade depolarization of the neuron, wherein the inhibitory proteindoes not have the sequence as set forth in GenBank accession numberEF474018 and uses an endogenous cofactor to produce the inhibitorycurrent.

According to another example embodiment of the present invention, amethod for generating an inhibitory-current flow in neurons isimplemented. The method includes in a neuron, engineering a protein thatresponds to light by producing an inhibitory current to dissuadedepolarization of the neuron, wherein the protein uses an endogenouscofactor and results in a toxicity of the engineered neuron that is lessthan about 75%.

According to another example embodiment of the present invention, amethod for controlling action potential of a neuron is implemented. Afirst light responsive protein is engineered in the neuron. The firstlight responsive protein does not have the sequence as set forth inGenBank accession number EF474018 and uses an endogenous cofactor toproduce the inhibitory current. In response to light, an inhibitorycurrent is produced in the neuron, the current generated from the firstlight responsive protein. A second light responsive protein isengineered in the neuron. In response to light, an excitation current isproduced in the neuron from the second light responsive protein.

According to another example embodiment of the present invention, amethod for controlling a voltage level across a cell membrane of a cellis implemented. A first light responsive protein is engineered in thecell. The voltage level across the cell membrane is measured. Light of afirst wavelength is generated in response to the measured voltage level.In response to light of a first wavelength and using the first lightresponsive protein, a first current is produced across the cell membranethat.

According to another example embodiment of the present invention, systemcontrols an action potential of a neuron in vivo. A delivery deviceintroduces a light responsive protein to the neuron, wherein the lightresponsive protein produces an inhibitory current and is not thesequence as set forth in GenBank accession number EF474018. A lightsource generates light for stimulating the light responsive protein. Acontrol device controls the generation of light by the light source.

According to another example embodiment of the present invention, amethod for treatment of a disorder is implemented. In a group of neuronsassociated with the disorder, inhibitory proteins are engineered thatuse an endogenous cofactor to respond to light by producing aninhibitory current to dissuade depolarization of the neurons, whereinthe engineered group of neurons has a toxicity of less than about 75%.The neurons are exposed to light, thereby dissuading depolarization ofthe neurons.

According to an example embodiment of the present invention, alight-responsive opsin is provided for use in therapy. The opsin can bea NpHR-based molecule for use in therapy wherein the molecule is capableof responding to light by producing an inhibitory current to dissuadedepolarization of a neuron and wherein the protein/molecule is capableof using an endogenous cofactor to produce the inhibitory current andmanifests a toxicity level that is less than 75%, at a high expressionlevel.

According to an example embodiment of the present invention, alight-responsive opsin is used in treating neurological diseases. Theopsin can include a nucleic acid molecule comprising a nucleotidesequence encoding a NpHR based protein for use in the treatment of CNSdisorders wherein said protein is capable of responding to light byproducing an inhibitory current to dissuade depolarization of a neuronusing an endogenous cofactor to produce the inhibitory current andmanifests a toxicity level that is less than 50%, at a high expressionlevel.

According to another example embodiment, a light-responsive opsin isused in the manufacture of a medicament for the treatment ofneurological diseases. For example, an NpHR-based protein in themanufacture of a medicament for the treatment of CNS disorders whereinthe said protein is capable of responding to light by producing aninhibitory current to dissuade depolarization of a neuron and is capableof using an endogenous cofactor to produce the inhibitory current andmanifests a toxicity level that is less than 75%, at a high expressionlevel.

According to another example embodiment, kit is provided foradministering treatment. The kit includes, for example a productcontaining a first light-responsive opsin and a second-light responsiveopsin as a combined preparation for simultaneous, separate or sequentialuse in the treatment of neurological diseases.

According to another example embodiment, a transgenic animal is producedwith a light-responsive opsin expressed in one or more cells.

According to another example embodiment, cells are modified, in a liveanimal, using light-responsive opsins. The animal is sacrificed and themodified cells are removed for study.

Other aspects and embodiments are directed to systems, methods, kits,compositions of matter and molecules for ion pumps or for controllinginhibitory currents in a cell (e.g., in in vivo and in vitroenvironments).

The above summary of the present invention is not intended to describeeach illustrated embodiment or every implementation of the presentinvention. The figures and detailed description that follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thedetailed description of various embodiments of the invention thatfollows in connection with the accompanying drawings, in which:

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N and 1O showexperimental results that are consistent with an example embodiment ofthe present invention;

FIGS. 2A, 2B, 2C, 2D, 2E and 2F show experimental results that areconsistent with an example embodiment of the present invention;

FIGS. 3A, 3B, 3C, 3D, 3E and 3F, show experimental results that areconsistent with an example embodiment of the present invention;

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G and 4H, show experimental results thatare consistent with an example embodiment of the present invention;

FIG. 5 shows a light source and modified cell, according to an exampleembodiment of the present invention;

FIG. 6 depicts an arrangement with multiple light sources, according toan example embodiment of the present invention;

FIG. 7 shows a system for controlling electrical properties of one ormore cells in vivo, according to an example embodiment of the presentinvention;

FIG. 8 shows a system for controlling electrical properties of one ormore cells in vivo, according to an example embodiment of the presentinvention, and

FIG. 9 shows Lentiviral vector construction, according to an exampleembodiment of the present invention.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION

The present invention is believed to be useful for facilitatingpractical application of a variety of photosensitive bio-molecularstructures, and the invention has been found to be particularly suitedfor use in arrangements and methods dealing with cellular membranevoltage control and stimulation. While the present invention is notnecessarily limited to such applications, various aspects of theinvention may be appreciated through a discussion of various examplesusing this context.

Consistent with one example embodiment of the present invention, alight-responsive protein/molecule is engineered in a cell. The proteinaffects a flow of ions across the cell membrane in response to light.This change in ion flow creates a corresponding change in the electricalproperties of the cells including, for example, the voltage and currentflow across the cell membrane. In one instance, the protein functions invivo using an endogenous cofactor to modify ion flow across the cellmembrane. In another instance, the protein changes the voltage acrossthe cell membrane so as to dissuade action potential firing in the cell.In yet another instance, the protein is capable of changing theelectrical properties of the cell within several milliseconds of thelight being introduced. For details on delivery of such proteins,reference may be made to U.S. patent application Ser. No. 11/459,636filed on Jul. 24, 2006 and entitled “Light-Activated Cation Channel andUses Thereof”, which is fully incorporated herein by reference.

Consistent with a more specific example embodiment of the presentinvention a protein, NpHR, from Natronomonas pharaonis is used fortemporally-precise optical inhibition of neural activity. NpHR allowsfor selective inhibition of single action potentials within rapid spiketrains and sustained blockade of spiking over many minutes. The actionspectrum of NpHR is strongly red-shifted relative to ChannelRhodopsin-2(ChR2) (derived from Chlamydomonas reinhardtii) but operates at similarlight power, and NpHR functions in mammals without exogenous cofactors.In one instance, both NpHR and ChR2 can be expressed in the targetcells. Likewise, NpHR and ChR2 can be targeted to C. elegans muscle andcholinergic motoneurons to control locomotion bidirectionally. In thisregard, NpHR and ChR2 form an optogenetic system for multimodal,high-speed, genetically-targeted, all-optical interrogation of livingneural circuits.

Certain aspects of the present invention are based on the identificationand development of an archaeal light-driven chloride pump, such ashalorhodopsin (NpHR), from Natronomonas pharaonis, fortemporally-precise optical inhibition of neural activity. The pumpallows both knockout of single action potentials within rapid spiketrains and sustained blockade of spiking over many minutes, and itoperates at similar light power compared to ChR2 but with a stronglyred-shifted action spectrum. The NpHR pump also functions in mammalswithout exogenous cofactors.

According to other example embodiments of the present invention, methodsfor generating an inhibitory neuron-current flow involve, in a neuron,engineering a protein that responds to light by producing an inhibitorycurrent to dissuade depolarization of the neuron. In one such method,the protein is halorhodopsin-based and in another method the protein isan inhibitory protein that uses an endogenous cofactor.

In another example embodiment, a method for controlling action potentialof a neuron involves the following steps: engineering a first lightresponsive protein in the neuron; producing, in response to light, aninhibitory current in the neuron and from the first light responsiveprotein; engineering a second light responsive protein in the neuron;and producing, in response to light, an excitation current in the neuronfrom the second light responsive protein.

Another embodiment involves method for controlling a voltage levelacross a cell membrane of a cell, the method includes: engineering afirst light responsive protein in the cell; measuring the voltage levelacross the cell membrane; and producing, in response to light of a firstwavelength and using the first light responsive protein, a currentacross the cell membrane that is responsive to the measured voltagelevel.

Another aspect of the present invention is directed to a system forcontrolling an action potential of a neuron in vivo. The system includesa delivery device, a light source, and a control device. The deliverydevice introduces a light responsive protein to the neuron, with thelight responsive protein producing an inhibitory current. The lightsource generates light for stimulating the light responsive protein, andthe control device controls the generation of light by the light source.

In more detailed embodiments, such a system is further adapted such thatthe delivery device introduces the light responsive protein by one oftransfection, transduction and microinjection, and/or such that thelight source introduces light to the neuron via one of an implantablelight generator and fiber-optics.

Another aspect of the present invention is directed to a method fortreatment of a disorder. The method targets a group of neuronsassociated with the disorder; and in this group, the method includesengineering an inhibitory protein that uses an endogenous cofactor torespond to light by producing an inhibitory current to dissuadedepolarization of the neurons, and exposing the neurons to light,thereby dissuading depolarization of the neurons.

According to yet another aspect of the present invention is directed toidentifying and developing an archaeal light-driven chloride pump, suchas halorhodopsin (NpHR), from Natronomonas pharaonic, fortemporally-precise optical inhibition of neural activity. The pumpallows both knockout of single action potentials within rapid spiketrains and sustained blockade of spiking over many minutes, and itoperates at similar light power compared to ChR2 but with a stronglyred-shifted action spectrum. The NpHR pump also functions in mammalswithout exogenous cofactors.

More detailed embodiments expand on such techniques. For instance,another aspect of the present invention co-expresses NpHR and ChR2 inthe species (e.g., a mouse and C. elegans). Also, NpHR and ChR2 areintegrated with calcium imaging in acute mammalian brain slices forbidirectional optical modulation and readout of neural activity.Likewise, NpHR and ChR2 can be targeted to C. elegans muscle andcholinergic motoneurons to control locomotion bidirectionally. TogetherNpHR and ChR2 can be used as a complete and complementary opto-geneticsystem for multimodal, high-speed, genetically-targeted, all-opticalinterrogation of living neural circuits.

In addition to NpHR and ChR2, there are a number of channelrhodopsins,halorhodopsins, and microbial opsins that can be engineered to opticallyregulate ion flux or second messengers within cells. Various embodimentsof the invention include codon-optimized, mutated, truncated, fusionproteins, targeted versions, or otherwise modified versions of such ionoptical regulators. Thus, ChR2 and NpHR (e.g., GenBank accession numberis EF474018 for the ‘mammalianized’ NpHR sequence and EF474017 for the‘mammalianized’ ChR2(1-315) sequence) are used as representative of anumber of different embodiments. Discussions specifically identifyingChR2 and NpHR are not meant to limit the invention to such specificexamples of optical regulators. For further details regarding the abovementioned sequences reference can be made to “Multimodal fast opticalinterrogation of neural circuitry” by Feng Zhang, et al, Nature (Apr. 5,2007) Vol. 446: 633-639, which is fully incorporated herein byreference.

Consistent with an example embodiment of the present invention, a methodis implemented for stimulating target cells in vivo using gene transfervectors (for example, viruses) capable of inducing photosensitive ionchannel growth (for example, ChR2 ion channels). The vectors can beimplanted in the body.

Consistent with a particular embodiment of the present invention, aprotein is introduced to one or more target cells. When introduced intoa cell, the protein changes the potential of the cell in response tolight having a certain frequency. This may result in a change in restingpotential that can be used to control (dissuade) action potentialfiring. In a specific example, the protein is a halorhodopsin that actsas a membrane pump for transferring charge across the cell membrane inresponse to light. Membrane pumps are energy transducers which useelectromagnetic or chemical bond energy for translocation of specificions across the membrane. For further information regardinghalorhodopsin membrane pumps reference can be made to “Halorhodopsin Isa Light-driven Chloride Pump” by Brigitte Schobert, et al, The Journalof Biological Chemistry Vol. 257, No. 17. Sep. 10, 1982, pp.10306-10313, which is fully incorporated herein by reference.

The protein dissuades firing of the action potential by moving thepotential of the cell away from the action potential trigger level forthe cell. In many neurons, this means that the protein increases thenegative voltage seen across the cell membrane. In a specific instance,the protein acts as a chloride ion pump that actively transfersnegatively charged chloride ions into the cell. In this manner, theprotein generates an inhibitory current across the cell membrane. Morespecifically, the protein responds to light by lowering the voltageacross the cell thereby decreasing the probability that an actionpotential or depolarization will occur.

As used herein, stimulation of a target cell is generally used todescribe modification of properties of the cell. For instance, thestimulus of a target cell may result in a change in the properties ofthe cell membrane that can lead to the depolarization or polarization ofthe target cell. In a particular instance, the target cell is a neuronand the stimulus affects the transmission of impulses by facilitating orinhibiting the generation of impulses by the neuron.

As discussed above, one embodiment of the present invention involves theuse of an optically responsive ion-pump that is expressed in a cell. Ina particular instance, the cell is either a neural cell or a stem cell.A specific embodiment involves in vivo animal cells expressing theion-pump. Certain aspects of the present invention are based on theidentification and development of an archaeal light-driven chloridepump, such as halorhodopsin (NpHR), from Natronomonas pharaonic, fortemporally-precise optical inhibition of neural activity. The pumpallows both knockout of single action potentials within rapid spiketrains and sustained blockade of spiking over many minutes, and itoperates at similar light power compared to ChR2 but with a stronglyred-shifted action spectrum. The NpHR pump also functions in mammalswithout exogenous cofactors.

According to an example embodiment of the present invention, anoptically responsive ion-pump and/or channel is expressed in one or morestem cells, progenitor cells, or progeny of stem or progenitor cells.Optical stimulation is used to activate expressed pumps/channels. Theactivation can be used to control the ion concentrations (e.g.,chloride, calcium, sodium, and potassium) in the cells. This can beparticularly useful for affecting the survival, proliferation,differentiation, de-differentiation, or lack of differentiation in thecells. Thus, optical stimulus is implemented to provide control over the(maturation) of stem or progenitor cells.

In a particular embodiment, optically-controlled stimulus patterns areapplied to the stem or progenitor cells over a period of hours or days.For further details regarding the effects of membrane potentials and ionconcentrations on such cells reference can be made to“Excitation-Neurogenesis Coupling in Adult Neural Stem/Progenitor Cells”by Karl Deisseroth, et al, Neuron (May 27, 2004) Neuron, Vol. 42,535-552 and to U.S. Patent Publication No. 20050267011 (U.S. patentapplication Ser. No. 11/134,720) entitled “Coupling of Excitation andNeurogenesis in Neural Stem/Progenitor Cells” to Deisseroth et al andfiled on May 19, 2005, which are each fully incorporated herein byreference.

In a particular embodiment, a method of driving differentiation in cellsis implemented. The cells are caused to express light-activatedNpHR-based protein. The cells are exposed to light to activate theNpHR-based protein. The activation drives differentiation of the exposedcell or the progeny of the exposed cell.

In another embodiment, the cells comprise stem cells.

Two exemplary ion pumps originate from two strains of archaea,Halobacterium salinarum (HsHR) and Natronomonas pharaonis (NpHR).Illumination of HsHR or NpHR-expressing oocytes leads to rapid outwardcurrents. Both HsHR and NpHR have excitation maxima near 580 nm as shownin FIG. 1A. Specifically, FIG. 1A shows the action spectrum of NpHR whenmeasured in Xenopus oocytes using a Xenon short arc lamp andnarrowbandwidth 20 nm filters, which is red-shifted from the known ChR2maximum of ˜460 nm. This spectral separation allows for ChR2 and an HRto be activated independently or in synchrony to effect bidirectionaloptical modulation of membrane potential.

In an experimental test, HsHR was found to have a lower extracellularCl− affinity than NpHR (Km,NpHR=16 mM in FIG. 1B, Km,HsHR=32 mM) andmeasured currents displayed rapid rundown at low extracellular [Cl−]that did not fully recover in darkness. The influence of cytoplasmic[Cl−] on HR pump currents was studied using excised giant patches. HRpump currents were not influenced by cytoplasmic [Cl−](from 0 to 124mM), indicating a very low affinity for Cl− on the cytoplasmic sidewhere Cl− ions are released, as expected since HR-mediated chloridepumping can achieve molar concentrations of cytoplasmic Cl−. The pumpcurrent exhibits more or less linear voltage dependence, and Cl− currentis robust for both HRs across all physiological voltage regimes.

In one instance, a mammalian codon-optimized NpHR gene fused withenhanced yellow fluorescent protein (NpHR-EYFP) was introduced intocultured rat hippocampal CA3/CA1 neurons using lentiviruses carrying theubiquitous EF-1α promoter (EF1 α::NpHR-EYFP). Cells expressing NpHR-EYFPexhibited robust expression for weeks after infection (FIG. 1C). Involtage clamp, illumination of NpHR-EYFP cells with yellow light(bandwidth 573-613 nm via Semrock filter FF01-593/40-25; 300 W xenonlamp) induced rapid outward currents (FIG. 1D, top) with a peak level of43.8±25.9 pA and a steady-state level of 36.4±24.4 pA (mean±s.d.reported throughout this paper, n=15; FIG. 1E). The relatively smalldifference between the peak and steady-state currents is believed to beindicative of rare deprotonation of the NpHR Schiff base during the pumpcycle 24. The rise time from light onset to 50% of the peak current isconsistent across all cells (6.0±1.0 ms; FIG. 1F) with rise and decaytime constants of Ton=6.1±2.1 ms and Toff=6.9±2.2 ms respectively.Light-evoked responses were never seen in cells expressing EYFP alone.In current clamp, NpHR-EYFP neurons exhibited light-evokedhyperpolarization (FIG. 1D; bottom) with an average peak of 14.7±6.9 mVand a steady-state of 12.1±6.6 mV (FIG. 1G). The delay from light onsetto 50% of hyperpolarization peak was 26.0±8.6 ms (FIG. 1F) and the riseand decay time constants were Ton=35.6±15.1 ms and Toff=40.5±25.3 msrespectively. To test whether NpHR-mediated hyperpolarization couldinhibit neuronal firing, current-clamped neurons were injected with a200 pA current step for 2 s to evoke robust spike firing; concurrentlight delivery abolished the evoked activity (FIG. 1H).

Images of NpHR-EYFP and ChR2-mCherry co-expressed in culturedhippocampal neurons were taken (FIG. 1I). NpHR function was probed usingcell-attached recordings with ChR2 photostimulation to drive reliablespike trains. Indeed, whereas trains of blue light pulses (see “blu” inFIG. 1J) were able to evoke action potentials, concomitant yellow lightillumination (see “yel” in FIG. 1J) abolished spike firing in bothcell-attached and subsequent whole-cell recoding modes (FIG. 1J). Afterachieving the whole-cell configuration, voltage-clamp recording showedthat independent exposure to yellow or blue light led to outward orinward photocurrents respectively (FIG. 1K), further confirming thatChR2 and NpHR can be combined to achieve bidirectional, independentlyaddressable modulation of membrane potential in the same neuron. Furtherconfirming that NpHR inhibitory function does not require a specificpipette chloride concentration under these recording conditions, it wasfound that NpHR-mediated inhibition is robust across a range of relevantwhole-cell pipette chloride concentrations (4-25 mM) and physiologicallynegative resting potentials, as expected from the fact that NpHR isdesigned to deliver chloride ions to molar levels in the archaealintracellular milieu.

Extensive controls were conducted to test whether heterologousexpression of NpHR in neurons would alter the membrane properties orsurvival of neurons. Lentiviral expression of NpHR for at least 2 weeksdid not alter neuronal resting potential (−53.1±6.3 mV for NpHR+ cells,−57.0±4.8 mV for NpHR− cells, and −56.7±5.7 mV for NpHR+ cells exposedto yellow light for 10 min followed by a delay period of 1 day; FIG. 1L,n=12 each) or membrane resistance (114.5±34.1 MΩ for NpHR+ cells,108.9±20.1 MΩ for NpHR− cells, and 111.4±23 MΩ for the light-exposedNpHR+ cells; FIG. 1M, n=12 each). These electrical measurementsindicated that NpHR has little basal electrical activity or passivecurrent-shunting ability and can be acceptable regarding cell health.

The dynamic electrical properties of neurons were tested with andwithout NpHR. There was no significant difference in the number ofspikes evoked by 500 ms current injection of 300 pA (7.5±2.8 for NpHR+neurons, 10.7±7.9 for NpHR− neurons, and 9.3±5.1 for the light-exposedNpHR+ neurons; FIG. 1N).

To assess cell survivability, both live NpHR+ neurons (with and withoutlight exposure) and NpHR− neurons were stained with themembrane-impermeant DNA-binding dye propidium iodide to assess cellsurvival. NpHR expression did not affect the percentage of neurons thattook up propidium iodide (13/240 for NpHR+ cells, 7/141 for NpHR− cells,and 10/205 for the light-exposed NpHR+ cells; FIG. 1M, P>0.999 by X²test). These experiments indicated that NpHR expression does notsignificantly affect the health or basal electrical properties ofneurons.

The tunability of NpHR efficacy with different intensities of deliveredlight was measured using a 200 pA current step that drove reliableaction potential trains. It was discovered that maximal light intensityof 21.7 mW/mm2 under a 40×, 0.8 NA water-immersion objective inhibited98.2±3.7% of the spikes.

Using a 200 pA current step to drive reliable action potential trains, amaximal light intensity of 21.7 mW/mm² under 40×, 0.8 NA water-immersionobjective inhibited 98.2±3.7% of spikes (FIGS. 2A and 2B). Using 33% or50% of the full light intensity inhibited 74.9±22.2% and 87.3±13.5% ofspikes, respectively (FIG. 2B). FIG. 2C shows that with steady currentinjection, lower intensities of light were effective for a shorterperiod of time; the delays from light onset to the first escaped spikeunder 33%, 50% and 100% light intensity were 533.65±388.2 ms,757.5±235.5 ms, and 990.5±19.1 ms, respectively. Therefore inhibition islikely to be more effective early in the light pulse, presumably due tothe slight inactivation of NpHR. Except where otherwise noted, theremaining experiments were conducted with 21.7 mW/mm2 yellow lightdelivered to the neurons.

Using trains of brief current pulses to generate spike trains, NpHR wastested for mediation of both long-term inhibition (to emulate lesions onthe timescale of seconds to minutes) and short-term inhibition (tomodify spike firing on the millisecond timescale). For long-terminhibition NpHR was tested over 10 min by injecting 300 pA currentpulses at 5 Hz to drive steady action potential firing. Concurrentyellow light was delivered continuously for 10 minutes. NpHR mediatedinhibition of spike trains remained effective over many minutes as shownby FIG. 2D. 99.0±1.9% of spikes were inhibited within the first twominutes while over 90% of spikes were inhibited for up to 8 minutes asshown in FIG. 2E, with n=5. The slight decrease in efficacy is likelydue to accumulation of non-functional NpHRs with a deprotonated Schiffbase over long periods of light exposure. While natural reprotonation ofthe Schiff base is slow, any non-functional NpHRs can be readily andquickly restored via brief illumination with blue light as shown by FIG.2F.

NpHR activation was tested for the ability to allow the “knockout” ofsingle action potentials. The fast photocurrent of ChR2 enables briefpulses of blue light to drive reliable action potential trains.Concurrently applied brief pulses of yellow light were used to testNpHR-mediated inhibition. FIG. 3A shows the results of an attempt toinhibit pairs of spikes in action potential trains of 5, 10, 20, and 30Hz. Indeed, single spikes could be reliably inhibited from within longerspike trains. Several pairs of spikes within a range of inter-spiketemporal delays were inhibited in an effort to define the temporalprecision of NpHR. FIGS. 3A, 3B and 3C show that both closely timed andtemporally separated spike pairs were able to be reliably inhibited,while sparing spikes between the targeted times (n=6). Over spike ratesof 5 to 30 Hz, the closely timed spikes could be selectively inhibitedwith a probability of 0.95 or greater. Moreover, FIG. 3D shows that bygiving trains of millisecond-scale yellow light pulses, it isstraightforward to simulate barrages of IPSP-like events with precise,reliable timing and amplitudes, from 5 to 100 Hz.

Since NpHR is a Cl− pump and not a channel, the light-driven inhibitionacts by shifting the membrane potential and will not contribute(significantly) to shunting or input resistance changes. FIGS. 3E and 3Fshow that, whereas the GABA_(A) chloride channel agonist muscimolsignificantly decreased neuronal input resistance, NpHR activation hadno detectable effect on the input resistance.

Since both ChR2 and NpHR can be activated with high temporal precisionusing millisecondscale blue or yellow light pulses, an experiment wasimplemented to test the possibility of driving both proteins inintermingled temporally precise patterns. Such ability can be useful tononinvasively activate or inhibit single identified action potentialswith light in the same experiment or even in the same cell. Cellattached and whole-cell recordings in hippocampal pyramidal neuronsrevealed that precisely patterned trains of yellow and blue light pulsescan be used to evoke and inhibit neural activity with single spikeprecision, and that NpHR can be used to override multiple preselectedChR2-driven spikes at identified positions in prolonged spike trains.

Both NpHR and ChR2 can be functionally expressed in the mammalian brainwithout exogenous delivery of its required cofactor all-trans-retinal(ATR), presumably due to the presence of endogenous retinoids in themammalian brain. As an experiment, lentiviruses carrying NpHR-EYFP weredelivered under the neuronal CaMKIIα promoter into the hippocampus ofthe adult mouse. Neurons throughout the hippocampus exhibited stableexpression of NpHR-EYFP, as indicated by a robust EYFP fluorescence.

NpHR-EYFP cells in acute hippocampal slices exhibited voltage clampphotocurrents similar to those observed in cultured neurons. A currentclamp recording of NpHR-EYFP neurons revealed that temporally precisepatterns of spike inhibition could be achieved readily as in dissociatedculture. No exogenous cofactors were delivered at any point, indicatingthat NpHR can be functionally applied to mammalian systems in vivo.

In another instance, NpHR/ChR2 was combined in a system by expressing inliving mammalian neural circuitry, with fura-2 calcium imaging, in anall-optical experiment. Lentiviruses carrying ChR2-mCherry under theneuron-specific CaMKIIα promoter and NpHR-EYFP under the EF-1 α promoterwere injected into the brain of postnatal d4 mouse pups; acute corticalslices were prepared at postnatal d10-14 and labeled with fura-2-AM. Inneurons co-expressing ChR2-mCherry and NpHR-EYFP, initial simultaneousillumination with both blue and yellow light did not lead to [Ca2+]transients while subsequent pulsed blue light alone in the same neuronsevoked ChR2-triggered [Ca2+] transients. This demonstrates that NpHR andChR2 can be integrated to achieve multimodal, bidirectional control ofneural activity in intact tissue. In the same imaged cells (where ChR2stimulation led to a 3.1±0.3% increase in ΔF/F), the combination of NpHRand ChR2 activation resulted in a 0.0±0.2% effect on ΔF/F (n=6,P<0.0001). Yellow illumination alone had no detectable effect on [Ca2+].Since not all targeted cells are necessarily affected to the samedegree, this optical system could complement electrophysiology to probesuccessful modulation of the targeted cell population. Thus, accordingto one embodiment, the combination of ChR2 and NpHR with calcium imagingprovides an all-optical system for interrogation of neural circuits.

Another set of experiments were conducted to show control of animalbehavior in vivo. An in vivo experiment involved expression of NpHR-ECFPfusion protein in the body wall muscles of the nematode Caenorhabditiselegans using the muscle-specific myosin promoter (Pmyo-3). ECFPfluorescence could be readily observed throughout muscle cells andmembranous muscle arm extensions. As worms (unlike mammals) appear notto have sufficient levels of endogenous retinoids, transgenic animalsexpressing NpHR in muscle were grown in medium containing ATR.Whole-cell voltage-clamp recordings from dissected muscles indeeddemonstrated light-evoked outward currents (265±82 pA, n=9). To testeffects on muscle activity, swimming behavior in liquid medium wasanalyzed. Consistent with the photocurrents observed, photoactivation ofNpHR immediately (within ˜150 ms) and essentially completely arrestedswimming behavior. Transgenic animals, raised in the absence of ATR andwild type animals raised with and without ATR, were used as controls.Robust paralyzing effects of light were observed, but consistently onlyin transgenic animals raised in the presence of ATR.

When transgenic muscle-expressing animals were illuminated for 1 second,they quickly returned to their natural swimming rate after lightstimulus termination. When NpHR was activated in muscle for 10 seconds,animals remained uncoordinated for prolonged periods (up to 40 seconds),before a full recovery became apparent and normal swimming commenced.

Another experiment involved targeting of NpHR to a specific class ofgenetically defined neurons in vivo. NpHR-ECFP was expressed incholinergic motoneurons using the vesicular acetylcholine transporterpromoter (Punc-17). When illuminated for 1 or 10 seconds, respectively,these animals also strongly reduced or essentially stopped swimmingbehavior. These animals, in contrast to the muscle targeted individuals,recovered to normal swimming behavior immediately, perhaps indicatingmore powerful Cl− homeostasis in neurons than in muscles, although inall cases full recovery was observed consistent with the lack oftoxicity observed in mammalian neurons. When illuminated on solid agarsubstrate, transgenic animals expressing NpHR either in muscle, or incholinergic motoneurons, exhibited rapid inhibition of movement andrelaxed their bodies, resulting in overall elongation by up to 9% within˜600 ms of illumination.

ChR2 and NpHR were found to be able to be driven simultaneously in C.elegans. With either muscle or targeted cholinergic neuron expression(using the Pmyo-3 or Punc-17 promoters, respectively), NpHR rapidly andreversibly counteracted the shortening behavior observed with ChR2alone. These experiments demonstrate that acetylcholine release can beefficiently triggered from C. elegans motoneurons using ChR2, and thatChR2 and NpHR work well together in nematodes as well as mammals. Insome instances, such an NpHR/ChR2 system enables rapid bidirectionalcontrol of neurons on the timescale of milliseconds, thus enablingemulation or alteration of the neural code. These fast genetically basedneural spike-controlling technologies powerfully augment existing toolsfor interrogating neural systems. Indeed, integration of the NpHR/ChR2neural control system with optical activity markers like fura-2, andwith GFP-based morphological markers, delivers a versatile triad oftechnologies for watching, listening to, and controlling living neuralcircuitry with light.

Both NpHR and ChR2 can be functionally expressed and operate at highspeed in the mammalian brain without necessitating cofactor addition.Moreover, NpHR and ChR2 function in behaving C. elegans as well aftersimple dietary ATR supplementation. When combined with optical imagingor behavioral measures in intact tissue or freely moving animals, theNpHR/ChR2 system provides the capability to directly and causally linkprecisely defined patterns of neural activity with specific circuitbehaviors.

The ability to use light to inhibit or activate neurons has practicalapplications beyond basic science investigations. The NpHR/ChR2 systemmay be genetically targeted to specific classes of neurons or otherexcitable cells involved in disease processes to enable highly preciseoptical therapeutic treatments. For example, in Parkinson's diseasewhere electrode-based deep brain stimulation (DBS) can be therapeuticfor symptomatic relief but also gives rise to side effects, delivery ofthese optogenetic tools targeted by cell type-specific promoters todistinct disease-related neuronal types may ultimately provide a moreprecise alternative with fewer side-effects. Whether in basic science orclinical applications, the spectral separation between the NpHR and ChR2activation maxima allows for the first time bidirectional opticalcontrol in the same target tissue, and permits both sufficiency andnecessity testing in elucidation of the roles of specific cell types inhigh-speed intact circuit function.

Oocyte microinjection and physiology were experimentally carried outusing the following procedures. NpHR cRNA was generated using the T7-capscribe kit from Ambion (Austin, Tex.). Stage V/VI oocytes were prepared.Each oocyte was injected with 30 to 50 ng cRNA, incubated for 4 to 7days at 16 to 18° C. with 1 μM ATR in the medium (90 mM NaCl, 2 mM KCl,1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.4/NaOH) to reconstitutefunctional HR. As a control uninjected oocytes were incubated in thesame medium. Oocytes were recorded using two-electrode voltage-clamp(Turbo Tec-05) and illuminated with a continuous He—Ne laser (594 nm,LYHR-0600M, Laser 2000, Wessling, Germany). The maximum light intensitywas 3 mW/mm² and was focused to a diameter close to the dimensions ofthe oocyte. In giant patch experiments from halorhodopsin-expressingoocytes a continuous He—Ne laser of 633 nm with light intensities up to400 mW/mm2 was used.

Lentiviral vector construction was experimentally carried out using thefollowing procedures. Lentiviral vectors containing SynapsinI::ChR2-mCherry, CaMKIIα::ChR2-mCherry, and CaMKIIα::NpHR-EYFP werebased on the FCK(1.3)GW plasmid. For the construction of theselentiviral vectors, the promoter was PCR amplified and cloned into thePacl and Agel restriction sites (FIG. 9). The transgene ChR2-mCherry orNpHR-EYFP were PCR amplified and cloned into the Agel and EcoRIrestriction sites. The pLEHYT vector is constructed in the same way aspLECYT3 by inserting the NpHR-EYFP gene into the Afel and Spelrestriction sites of pLEGT.

For both NpHR-EYFP and ChR2-mCherry, the protein fusion was made via aNotI restriction site. The linker between the two proteins is5′-GCGGCCGCC-3′. The start codon on the fluorescent protein was removeddeliberately to avoid translation of the fluorescent protein alone. Inaddition to the promoter, each lentiviral vector contains the HIV-1central polypurine tract (cPPT) and the Woodchuck Hepatitis VirusPost-transcriptional Regulatory Element (WPRE) to improve transductionefficiency.

Lentiviral production and transduction were experimentally carried outusing the following procedures. High-titer lentiviruses were producedusing a second generation lentiviral system, by cotransfection of 293FTcells (Invitrogen) with pCMVΔR8.74 and pMD2.G in addition to the viralvector. The following protocol was used.

Day 0: Split 4 T-225 flasks (Nunc) of 95% confluent 293FT cells into one4 layer CellFactory (Nunc). Culture using 500 mL of DMEM with 10% FBS.Incubate the plates at 37° C. overnight. The cells should reach 90%confluence in 24 hours.

Day 1: Perform calcium phosphate transfection; make DNA mixturecontaining 690 μg of the viral vector, 690 μg of pCMVΔR8.74, and 460 μgof pMD2.G. Add 5.7 mL of 2M CaCl2 to the DNA mixture and bring the totalvolume to 23.75 mL with distilled H2O; then, quickly combine theDNA/CaCl2 solution with 23.75 mL of 2×HBS (50 mM HEPES, 1.5 mM Na2HPO4,180 mM NaCl, pH 7.05; note that the pH is important); after quicklymixing by inverting 5 times, add the DNA/CaCl2/HBS solution to 500 mL ofroom-temperature DMEM with 10% FBS to make the transfection media; then,exchange the media in the CellFactory with the transfection media.

Day 2: 15 hours from the time of transfection, remove the transfectionmedia from the CellFactory and wash the cells 3 times with freshroom-temperature DMEM; incubation longer than 15 hours may lead to celldeath and reduced viral titer; finally, replace the media with 500 mL offresh DMEM containing 10% FBS and incubate in a 37° C. incubator for 9hours.

Day 2.5: 24 hours from the time of transfection, remove the media fromthe CellFactory and replace with 200 mL of serum-free media(UltraCULTURE, Cambrex) containing 5 mM Sodium Butyrate; return theCellFactory to the incubator.

Day 3: 40 hours from the time of transfection, collect the 200 mL ofmedia from the CellFactory. This is the viral-containing supernatant.Centrifuge at 1000 rpm for 5 minutes to precipitate large cell debrisand then filter the viral supernatant using a 0.45 μm low-proteinbinding filter flask. Then, centrifuge the supernatant using a SW-28rotor (Beckman Coulter) for 2 hours at 55,000×g to precipitate thevirus. Usually 6 centrifuge tubes are required to concentrate all of theviral supernatant. Before spinning, add 2 mL of PBS containing 20%sucrose to the bottom of the centrifuge tube to remove any remainingcell debris during centrifugation. After centrifugation, gently decantthe liquid from the centrifuge tubes and re-suspend all 6 viral pelletswith 100 μL of 4° C. PBS. Then, aliquot the viral solution and store at−80° C. for future use. If desired, 10 mL of unconcentrated viralsupernatant can be stored before centrifugation for in vitro use incultured neurons. For culture applications, neurons can be transducedsimply by adding 50 μL of unconcentrated viral supernatant per 24-wellplate well. Protein expression can be observed 4 to 5 days later. For invivo applications, concentrated virus can be directly injected into themammalian brain.

For whole-cell and cell-attached recording in cultured hippocampalneurons or acute brain slices, three intracellular solutions containing4 mM chloride were prepared (135 mM K-Gluconate, 10 mM HEPES, 4 mM KCl,4 mM MgATP, 0.3 mM Na3GTP, titrated to pH 7.2), 10 mM chloride (129 mMK-Gluconate, 10 mM HEPES, 10 mM KCl, 4 mM MgATP, 0.3 mM Na3GTP, titratedto pH 7.2), or 25 mM chloride (114 mM K-Gluconate, 10 mM HEPES, 25 mMKCl, 4 mM MgATP, 0.3 mM Na3GTP, titrated to pH 7.2).

For cultured hippocampal neurons, Tyrode's solution was employed as theextracellular solution (125 mM NaCl, 2 mM KCl, 3 mM CaCl2, 1 mM MgCl2,30 mM glucose, and 25 mM HEPE, titrated to pH 7.3).

For preparation of acute brain slices, mice were sacrificed 2 weeksafter viral injection. 250 μm acute brain slices were prepared inice-cold cutting solution (64 mM NaCl, 25 mM NaHCO3, 10 mM glucose, 120mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 7 mM MgCl2, andequilibrated with 95% O2/5% CO2) using a vibratome (VT1000S, Leica).Slices were incubated in oxygenated ACSF (124 mM NaCl, 3 mM KCl, 26 mMNaHCO3, 1.25 mM NaH2PO4, 2.4 mM CaCl2, 1.3 mM MgCl2, 10 mM glucose, andequilibrated with 95%+O2/5% CO2) at 32° C. for 30 min to recover.

For calcium imaging, lentiviruses were injected into the cortex ofC57BL/6 mice at postnatal day 4 or 5 and acute brain slices wereprepared 7 to 8 days later for adult mice.

Transgenic C. elegans Lines and Transgenes were experimentally developedusing the following procedures. The NpHR gene was placed under themuscle-specific myo-3 promoter (untagged NpHR in transgenezxEx29[pmyo-3::NpHR; lin-15+] and NpHR-ECFP in transgenezxEx30[pmyo-3::NpHR-ECFP; rol-6d]) or under the cholinergic motoneuronspecific unc-17 promoter (NpHRECFP in transgenezxEx33[punc-17::NpHR-ECFP; lin-15+]). The NpHR-ECFP fusion (zxEx30 andzxEx34, see below) was employed to assess expression pattern. NpHR-ECFP(zxEx30) animals showed light induced effects that were comparable tothe untagged version (zxEx29).

For co-activation of ChR2/NpHR in muscles or cholinergic motoneurons,transgenes zxEx32[pmyo-3::NpHR; pmyo-3::ChR2(H134R)-EYFP; lin-15+] andzxEx34[punc-17::NpHRECFP; punc-17::ChR2(gf)-YFP; rol-6d] were used.Table 1 lists examples of transgenes and worm lines used in variousexperimental tests.

TABLE 1 Transgene Genotype Strain zxEx29 [p myo-3::NpHR; lin-15⁺] lin-15(n765ts⁻) ZX396 zxEx30 [p myo-3::NpHR-ECFP; rol-6d] N2 ZX397 zxEx32 [pmyo-3::NpHR; lin-15 (n765ts⁻) ZX399 p myo-3::ChR2(H134R)-EYFP; lin-15⁺]zxEx33 [p unc-17::NpHR-ECFP; lin-15⁺] lin-15 (n765ts⁻) ZX416 zxEx34 [punc-17::NpHR-ECFP; p unc-17:: N2 ZX417 ChR2(H134R)-EYFP; rol-6d]

Experimental tests for bidirectional optical neural control and in vivoimplementation were implemented. NpHR in muscle were grown in mediumcontaining ATR. Whole-cell voltage-clamp recordings from dissectedmuscles indeed demonstrated light-evoked outward currents (265±82 pA,n=9). To test effects on muscle activity, swimming behavior in liquidmedium was analyzed. Consistent with the photocurrents observed,photoactivation of NpHR immediately (within ˜150 ms) and essentiallycompletely arrested swimming behavior. As controls, transgenic animalsthat were raised in the absence of ATR, and wild type animals that wereraised with and without ATR were used. Robust paralyzing effects oflight were observed, but consistently only in transgenic animals raisedin the presence of ATR. When muscle-expressing animals were illuminatedfor 1 s, they quickly returned to their natural swimming rate afterlight stimulus termination. When NpHR was activated in muscle for 10 s,animals remained uncoordinated for prolonged periods (up to 40 seconds),before a full recovery became apparent and normal swimming commenced.

Next, NpHR was targeted to a specific class of genetically definedneurons in vivo. NpHR-ECFP was expressed in cholinergic motoneuronsusing the vesicular acetylcholine transporter promoter (Punc-17). Whenilluminated for 1 or 10 s, respectively, these animals also stronglyreduced or essentially stopped swimming behavior. These animals, incontrast to the muscle-targeted individuals, recovered to normalswimming behavior immediately, perhaps indicating more powerful Cl⁻homeostasis in neurons than in muscles, although in all cases fullrecovery was observed consistent with the lack of toxicity observed inmammalian neurons.

When illuminated on solid agar substrate, transgenic animals expressingNpHR either in muscle, or in cholinergic motoneurons, exhibited rapidinhibition of movement and relaxed their bodies, resulting in overallelongation by up to 9% within ˜600 ms of illumination. It was found thatChR2 and NpHR could be driven simultaneously in C. elegans as well. Witheither muscle or targeted cholinergic neuron expression (using thePmyo-3 or Punc-17 promoters, respectively), NpHR rapidly and reversiblycounteracted the shortening behavior observed with ChR2 alone. Theseexperiments demonstrate that acetylcholine release can be efficientlytriggered from C. elegans motoneurons using ChR2, and that ChR2 and NpHRwork well together in nematodes as well as mammals.

Slides were developed showing cell-attached (FIG. 4A left) andwhole-cell (FIG. 4A right) recording of cultured hippocampal neuronsco-expressing ChR2-mCherry and NpHR-EYFP. Action potentials were evokedvia trains of blue light pulses (5 Hz trains, 15 ms pulse width, lowestset of bars). NpHR-mediated inhibition was co-administered by briefyellow light pulses (50 ms pulse width, upper sets of bars).

A confocal image was taken from acute mouse brain slice showingmembrane-localized NpHR-EYFP expression in the hippocampal CA3 subfield(FIG. 4B left). Current clamp recording showed NpHR-mediated inhibitionof specific spikes during a train of action potentials evoked by pulsedcurrent injection (300 pA, 20 Hz, FIG. 4B right).

Epifluorescence images of cortical neurons triple-labeled withNpHR-EYFP, ChR2-mCherry, and Fura-2 showed expression in the neurons.(FIG. 4C)

Simultaneous illumination of cells co-expressing NpHR-EYFP andChR2-mCherry with steady yellow (continuous illumination, 6 s) andpulsed blue light (50 pulses at 15 ms per flash, 10 Hz) prevented [Ca2+]transients (FIG. 4D). Subsequent photostimulation of the same cells withblue light pulses (50 pulses at 15 ms per flash, 10 Hz) evoked reliable[Ca2+] transients. A bar graph was generated that shows thephotostimulation-induced fluorescence changes (FIG. 4D, n=6ChR2-activated triple-labeled cells).

Epifluorescence images showed Pmyo-3-mediated (transgene zxEx30)NpHR-ECFP expression in the body wall muscles of C. elegans (FIG. 4E). Amuscle voltage clamp trace showed photocurrent in transgenic C. elegansexpressing NpHR-ECFP (transgene zxEx30) and raised in the presence ofATR. A lack of response in transgenic animal raised in the absence ofATR was noted. Animal postures from three consecutive movie frames(frame rate 12.5 Hz), either with or without NpHR photoactivation, weresuperimposed to show lack of movement in NpHR photoactivated animals.

The effect of 10 s illumination on swimming rate (n=10 for each set) inwild type controls, animals expressing NpHR in muscles (transgenezxEx29, FIG. 4F blue), or cholinergic motoneurons (transgene zxEx33,FIG. 4F red) was monitored. The number of swimming cycles per second wascounted in bins of 5 s intervals. A briefer 1 s illumination protocolwas also used, revealing rapid inhibition during illumination and fasterrecovery by comparison with the 10 s illumination.

The effect of 1 s illumination on body length (n=5-6 for each set) wasmonitored (FIG. 4G). Movies were taken from transgenic worms expressingNpHR in muscles (transgene zxEx29) or cholinergic motoneurons (transgenezxEx33). Combined ChR2/NpHR expression in muscle cells (transgenezxEx32) or cholinergic motoneurons (transgene zxEx34) of behaving worms.A plot was generated that shows the body length during the first framebefore illumination, the 13 frames during the illumination and the next11 frames after illumination ended (FIGS. 4F, 4G and 4H) includingcontrols and combined ChR2/NpHR expression in muscle cells (transgenezxEc32) or cholinergic motoneurons (transgene zxEx34) of behaving worms.

NpHR activation significantly reversed the muscle contraction caused byChR2 activation (n=6 per condition; *, p<0.05; **, p<0.01; ***, p<0.001;between consecutive time points; t-test), but not in animals raised inthe absence of ATR.

According to a first experimental method, swimming of a transgenic C.elegans expressing NpHR (transgene zxEx29) in muscles isinstantaneously, and repeatedly, inhibited by photoactivation of HR.

According to a second experimental method, swimming of a transgenic C.elegans expressing NpHR in cholinergic motoneurons (transgene zxEx33) isinstantaneously inhibited by photoactivation of NpHR.

According to a third experimental method, transgenic C. elegansexpressing NpHR-ECFP in muscles (transgene zxEx30). Movement is rapidlyinhibited (3×) by photoactivation of HR, and the body relaxes anddilates.

According to a fourth experimental method, one transgenic C. elegansexpressing NpHR in muscles (transgene zxEx29), and one non-transgeniccontrol animal. Movement of the transgenic animal is rapidly inhibitedby photoactivation of HR.

According to a fifth experimental method, transgenic C. elegansexpressing NpHR in cholinergic motoneurons (transgene zxEx33). Movementis rapidly inhibited by photoactivation of HR, and the body relaxes anddilates.

According to a sixth experimental method, co-expression and -activationof ChR2(H134R)-EYFP and NpHR in cholinergic motoneurons of transgenic C.elegans (transgene zxEx34). The animal is illuminated with blue lightfor ChR2 activation, causing contractions, then, while ChR2 is stillphotoactivated, NpHR is photoactivated by yellow light, causingsignificant body relaxation. When NpHR activation ends, the animalcontracts again (ChR2 still activated), and finally, when ChR2activation ends, the animal relaxes to the initial body length.

According to a seventh experimental method, co-expression and rapidlyalternating activation of ChR2(H134R)EYFP and NpHR in muscles oftransgenic C. elegans (transgene zxEx32). The animal is illuminated withalternating blue light (for ChR2 activation), causing contractions, andyellow light causing significant body relaxation.

FIG. 5 depicts an arrangement with multiple light sources, according toan example embodiment of the present invention. FIG. 5 shows lightsources 502 and 504 that illuminate proteins 510 and 514. The proteins510 and 514 are engineered within cell 512 to control current across thecell membrane in response to light from light sources 502 and 504,respectively. In one instance, the first protein 510 functions todissuade action potential firing, while the second protein 514 functionsto encourage action potential firing. Each of proteins 510 and 514 areresponsive to light. In a particular instance, the first protein isresponsive to light from light source 502 having a wavelength A and thesecond protein is responsive to light from light source 504 having awavelength B. Thus, the light sources can be used to control eachprotein independently. This can be useful for both encouraging anddissuading action potentials in the cell. In another instance, havingboth types of proteins allows for both positive and negative control ofthe cell membrane voltage. Thus, the different light sources andproteins could be used to control the voltage or current level (e.g.,clamping) of the cell membrane.

One method of determining responsiveness involves quantifying theresponsiveness in terms of the intensity of light required to produce agiven response. In some instances, the first or second protein can stillbe responsive to the alternate wavelength of light although theresponsiveness of the protein may be less than that of the primarywavelength. Accordingly, a protein of a first type may have someresponsiveness to the wavelength corresponding to the other type ofprotein while still maintaining sufficient independence of operation. Inone such instance, control of the cell can be implemented by shiftingeither the wavelength of light or the intensity of the light. Forinstance, the wavelength can be shifted between A and B to induce acorresponding increase or decrease the membrane voltage potential.

Embodiments of the invention can be implemented with just the proteinbased ion pump(s). In a specific example, pump 510 is designed tooperate using an endogenous cofactor, such as ATR, which can be found inpeople and many animals. This is particularly useful for minimizingintrusiveness of in vivo applications because it can reduce the need forforeign substances (e.g., cofactors). In a particular instance, pump isa halorhodopsin that acts as an anion pump (e.g., Cl⁻) that is activatedin response to light from light source 502 within milliseconds. Such afast response allows for the system to control (dissuade) individualaction potentials in the cell.

According to one embodiment of the present invention, pump 514 canoptionally be implemented for purposes other than dissuading actionpotential firing, such as controlling the voltage level of cell 508.More specifically, a sensor can be used to provide feedback to the lightsource 502. For instance, this feedback could be a measurement of thevoltage or current across the cell membrane. Thus, the light sourcecould be configured to maintain a constant current or voltage (e.g.,clamp) across the cell. Moreover, the amount of responsiveness can becontrolled by modifying one or more of the intensity and wavelength ofthe light.

FIG. 6 shows a system for controlling electrical properties of one ormore cells in vivo, according to an example embodiment of the presentinvention. Control/Interface unit 602 enables/disables light source 604to illuminate target cells 608. A delivery mechanism, such as fiberoptic cable 606, routes or otherwise directs the light to target cells608. Fiber optic cable 606 may include a bundle of optical cables, eachcapable of carrying and directing light independently. Thus, fiber opticcable 606 can be configured to deliver light having one or morewavelengths to multiple locations. Sensor 610 can be implemented, e.g.,as an optical device such as an optical scope or as a voltmeter, toprovide feedback to control unit 642. In a particular instance, thefeedback includes optical imaging of the target cells or of otherrelated cells. In another instance, the feedback could monitor thevoltage response of the target cells, including the amount of actionpotential firing.

FIG. 7 shows a system for controlling electrical properties of one ormore cells in vivo, according to an example embodiment of the presentinvention. Control/Interface unit 702 enables/disables implantable lightsource 704, which in turn illuminates target cells 706. Light source 704is shown with two light source, inhibitory current light source 708 andexcitation current light source 710. Light source 708 produces light ata wavelength and intensity that an inhibitory protein is responsive to,while light source 710 produces light at a wavelength and intensity thatan excitation protein is responsive to. One skilled in the art wouldrecognize that various configurations of light source 710 are possible,including a single inhibitory light source or an array of light sourceshaving one or more wavelengths. Control/Interface unit 702 communicateswith light source 704 through any suitable communication mechanisms,such as wired communications or wireless communications usingradio-frequency signals, magnetic signals and the like. As discussedabove in connection with FIG. 6, sensor 712 can optionally beimplemented for providing feedback to control unit 702.

Another important aspect of the present invention concerns applicationsand uses which benefit by reducing toxicity of cells. In certainapplications and uses, cells modified to include ion pump molecules canbecome intolerably toxic. Toxicity can become increasingly problematicas the expression level increases in a cell or the network, for example,when expecting consistent results under repeated tests using the samecell or neural network. A number of embodiments discussed abovespecifically mention the mammalianized NpHR sequence (GenBank AccessionNo. EF474018). In connection with the present invention, it has beendiscovered that this mammalianized NpHR coding sequence has a toxicitythat is nearly 100%. It has been discovered that for high expressionlevels the mammalianized NpHR sequence manifests toxicity at 87% whichis considered near enough complete toxicity to be considered as abaseline toxicity reference level which can be substantially reducedusing different (NpHR-based but having a different NpHR-based sequence)molecules. Accordingly, various aspects of the present invention areimplemented with significantly reduced toxicity.

The toxicity levels were obtained by a stringent process of identifyingany cell abnormality, such as blebs on the cell membrane. For thepurposes of this disclosure and the data presented herein, a cell witheven single abnormality is considered toxic as are any dead cells. A“toxicity level,” as used in this disclosure, means the percentage ofcells that are considered toxic.

As discussed herein, the expression levels are obtained by increasingthe original expression levels discussed above (i.e., about 2×10e7infectious units (ifu) per milliliter) to one of high expression of atleast 3×10e7 ifu per milliliter; very high expression of at least 4×10e7ifu per milliliter; ultra high expression of at least 5×10e7 ifu permilliliter; or very ultra high expression of at least 1×10e8 ifu permilliliter. In a particular embodiment the expression levels cancharacterized in terms of their mean photo current being 44 pA orhigher.

Various embodiments of the present invention allow for substantiallyreduced toxicity levels in high expression level (relative to themammalianized NpHR sequence).

According to one embodiment, an opsin sequence is implemented thatexhibits a toxicity of about 72%. A specific example is SPChR2-NpHR (SEQID NO 7), where a signal peptide (first 15aa) from ChR2 is added to theN-terminus (SEQ ID NO 11): DYGGALSAVGRELL.

According to one embodiment, an opsin sequence is implemented thatexhibits a toxicity of about 69%. A specific example is SPnAChR-L-NpHR(SEQ ID NO 8), where signal peptide (23aa) from nicotinic acetylcholinereceptor is added to the N-terminus (SEQ ID NO 12):MGLRALMLWLLAAAGLVRESLQG.

According to one embodiment, an opsin sequence is implemented thatexhibits a toxicity of about 59%. A specific example is NpHR-VSNL (SEQID NO 9), where the PDZ binding motif (SEQ ID NO 24) VSNL is added tothe C-terminus.

According to one embodiment, an NpHR coding sequence is implemented witha toxicity of about half of the base-line toxicity. A specific exampleis an NpHR-based sequence that uses the codons originally present inNatronomonas pharaonis. Another example is an NpHR-based sequence wherea signal peptide (18aa) from nicotinic acetylcholine receptor is addedto the N-terminus (SEQ ID NO 13): MRGTPLLLVVSLFSLLQD.

According to another embodiment, an NpHR coding sequence is implementedwith a toxicity of between about 34% and 40%. A specific example is anNpHR-based sequence that is formed by adding the PDZ binding motif (SEQID NO 14) ETQZ to the C-terminus (NpHR-ETQV).

According to another embodiment, an NpHR coding sequence is implementedwith a toxicity of between about 20% and 26%. A specific example is anNpHR-based sequence that is formed by adding the (SEQ ID NO 15) PTPPsequence to the C-terminus to promote interaction with actin-bindingprotein filamin (NpHR-actin).

According to another embodiment, an NpHR coding sequence is implementedwith a toxicity of between about 4% and 10%. A specific example is anNpHR-based sequence that is formed by adding the ER export signal to theC-terminus: (SEQ ID NO 16) VLGSL or, more generally, (SEQ ID NO 17)VXXSL (NpHR-ERexport).

According to another embodiment, an NpHR coding sequence is implementedwith a peak current of between about 11.1 pA and 50 pA. A specificexample is an NpHR-based sequence that uses the codons originallypresent in Natronomonas pharaonis.

According to another embodiment, an NpHR coding sequence is implementedwith a peak current of between about 40 pA and 61 pA. A specific exampleis an NpHR-based sequence where a signal peptide (18aa) from nicotinicacetylcholine receptor is added to the N-terminus: (SEQ ID NO 13)MRGTPLLLVVSLFSLLQD.

According to another embodiment, an NpHR coding sequence is implementedwith a peak current of between about 49 pA and 77 pA. A specific exampleis an NpHR-based sequence that is formed by adding the PDZ binding motif(SEQ ID NO 14) ETQZ to the C-terminus (NpHR-ETQV).

According to another embodiment, an NpHR coding sequence is implementedwith a peak current of between about 11 pA and 70 pA. A specific exampleis an NpHR-based sequence that is formed by adding the (SEQ ID NO 15)PTPP sequence to the C-terminus to promote interaction withactin-binding protein filamin (NpHR-actin). Another example is anNpHR-based sequence that is formed by adding the ER export signal to theC-terminus: VLGSL or, more generally, (SEQ ID NO 17) VXXSL(NpHR-ERexport).

Experimental data results showing toxicity and peak current for variousNpHR sequences are shown in Table 2.

TABLE 2 Toxicity Peak Molecular in photocurrent Construct Modificationneurons (mean ± s.d) Humanized none 87% 48.0 ± 7.0 pA  NpHR (SEQ IDNO 1) Non- use the original codons  37% 29.6 ± 18.5 pA Humanizedfrom bacteria NpHR (SEQ ID NO 2) SP_(nAChR-S)-Added signal peptide (18aa) 37% 51.5 ± 9.2 pA  NpHRfrom nicotinic acetylcholine (SEQ ID receptor to the N-terminus: NO 3)MRGTPLLLVVSLFSLLQD NpHR- Added the PDZ binding 34% 63.7 ± 12.7 pA ETQVmotif ETQV to the C- (SEQ ID terminus NO 4) NpHR-actinAdded the PTPP sequence to 23% 39.8 ± 20.2 pA (SEQ IDthe C-terminus to promote NO 5) interaction with actin-bindingprotein filamin NpHR- Added the ER export signal  7% 40.3 ± 28.5 pAERexport to the C-terminus: VLGSL (SEQ ID (more general VXXSL) NO 6)

Toxicity was assessed in cultured hippocampal neurons as follows:neurons 4 days in vitro were infected with lentivirus for each of theconstructs and allowed to accumulate protein for two weeks beforeassessing toxicity. Cells that displayed large round intracellular blobseither in the soma or dendrites were counted as toxic cells. Thephotocurrents for each of the constructs were assessed byelectrophysiology as described herein and also similar to that taught by“Multimodal fast optical interrogation of neural circuitry” by Zhang, etal, Nature (Apr. 5, 2007) Vol. 446: 633-639, which is fully incorporatedherein by reference. It should be noted that while the neurons wereallowed to express protein for at least one week, this time frame wasabout half of the time allotted in various underlying experimental testsdiscussed above. Additional expression time would allow for moreexpression, which in turn would result in increased toxicity andphoto-currents. Accordingly, assuming a near-linear increase intoxicity, the embodiments showing around 37% toxicity would reach about74%.

Various embodiments are directed toward a construct that includes ERexport signals, including, but not limited to: (SEQ ID NO 17) VXXSL;(SEQ ID NO 18) FXYENE (see “A sequence motif responsible for ER exportand surface expression of Kir2.0 inward rectifier K(+) channels”Stockklausner et al., FEBS Lett.; 493 (2-3):129-133 March, 2001; and“Role of ER Export Signals in Controlling Surface Potassium ChannelNumbers” Ma et al., Science Vol. 291. no. 5502:316-319, 2001);C-terminal valine residue (see “A Specific Endoplasmic Reticulum ExportSignal Drives Transport of Stem Cell Factor (Kitl) to the Cell Surface”Paulhe et al., J. Biol. Chem., Vol. 279, Issue 53, 55545-55555, Dec. 31,2004); VMI (see “Signal-dependent export of GABA transporter 1 from theER-Golgi intermediate compartment is specified by a C-terminal motif”Farhan et al., J. Cell Sci. 121:753-761, Feb. 19, 2008.) Each of theabove-mentioned references is incorporated herein by reference in theirentirety.

Various embodiments are directed toward a construct that includes signalpeptides for insertion into plasma membrane including, but not limitedto, signal peptides from other opsins or from other transmembraneproteins such as the nicotinic acetylcholine receptor (Isenberg et al.,1989, J. Neurochemistry).

For additional information regarding implementation of a signal peptide(first 15aa) from ChR2 added to the N-terminus reference can be made to“Millisecond-timescale, genetically-targeted optical control of neuralactivity” Boyden, et al., Nature Neuroscience 8(9):1263-1268 (2005),which is fully incorporated herein by reference.

For additional information regarding implementation of a signal peptide(23aa) from nicotinic acetylcholine receptor added to the N-terminusreference can be made to Bocquet et al., “A prokaryotic proton-gated ionchannel from the nicotinic acetylcholine receptor family” Nature445:116-119, January, 2007.

Various embodiments are directed toward a construct that includes PDZbinding motifs including, but not limited to, (SEQ ID NOS 19-20)X(S/T)XV (SEQ ID NO 21) ETQV) or (SEQ ID NOS 22-23) X(S/T)XL (e.g.,VSNL).

For additional information regarding implementation of NpHR-VSNL, wherethe PDZ binding motif VSNL is added to the C-terminus, reference can bemade to “Interactions with PDZ proteins are required for L-type calciumchannels to activate cAMP response element-binding protein-dependentgene expression” Weick et al., J. Neurosci. 23:3446-3456, 2003, which isfully incorporated herein by reference.

For additional information regarding implementation of NpHR-ETQV-basedsequences, reference can be made to “Targeting and Readout Strategiesfor Fast Optical Neural Control In Vitro and In Vivo” Gradinaru, et al.,The Journal of Neuroscience 27(52):14231-14238, Dec. 26, 2007, which isfully incorporated herein by reference.

For additional information regarding implementation of originalcodon-based sequences, reference can be made to “Light Driven ProtonPump or Chloride Pump by Halorhodopsin” Bamberg, et al., Proc. Natl.Acad. Sci. USA Vol. 90: 639-643, January 1993 Biophysics, which is fullyincorporated herein by reference.

For additional information regarding implementation ofSP_(nAchR-S)-pHR-based sequences, reference can be made to “RapidCommunication Cloning of a Putative Neuronal Nicotinic AcetylcholineReceptor Subunit” Isenberg, et al., J. Neurochem. 52(3):988-991, 1989,which is fully incorporated herein by reference.

For additional information regarding implementation of NpHR-actin-basedsequences, reference can be made to “Localization and Enhanced CurrentDensity of the Kv4.2 Potassium Channel by Interaction with theActin-binding Protein Filamin” Petrecca, et al., The Journal ofNeuroscience 20(23):8763-8744, December, 2000, which is fullyincorporated herein by reference.

For additional information regarding implementation of ERexport-basedsequences, reference can be made to “Surface Expression of Kv1Voltage-Gated K+ Channels is Governed by a C-terminal Motif” Levitan, etal., TCM 10(7):317-320, 2000, which is fully incorporated herein byreference.

The various sequences need not be implemented in isolation. To thecontrary, embodiments of the present invention involve variouscombinations of the above sequences with one another on the same gene.For example, the desired expression levels, peak currents and toxicitylevels are tailored through a combination of two or more differentsequences within the target cellular structure(s). Specific embodimentsinvolve the selection and use of specific sequences that bind todifferent portions of the gene (e.g., the C-terminus and theN-terminus). Such selections results in characteristics (e.g., toxicity,current levels and expression levels) not present in embodiments thatuse only a single sequence. Thus, various embodiments are directedtoward combinations of two or more of the above listed modifications.For example, a hybrid construct of SP_(nAChR-S)-NpHR-ERexport (SEQ ID NO10). Another example involves the addition of ChR2 to the cell.

Thus, various embodiments can be implemented in which two or moreconstructs are used to express an NpHR-based protein in the cell. Eachconstruct is capable of independently expressing the protein. In oneinstance, the constructs can be implemented in the same sequence. Inanother instance, the constructs can be sequentially delivered using twoor more different sequences.

Additional constructs can be implemented by co-expressing helperproteins, such as chaperones, that could be useful in aiding NpHRfolding and trafficking.

A few example sequence listings are provided in the Appendix, whichforms part of this specification. It should be noted that all thesequences contain the EYFP fluorescent protein for visualizationpurposes. Embodiments of the present invention can be implementedwithout this EYFP fluorescent protein. For example, each of thesequences can be implemented either on the NpHR alone (with nofluorescent protein) or on the NpHR-X fluorescent protein complex.

Many human applications of the present invention require governmentalapproval prior to their use. For instance, human use of gene therapy mayrequire such approval. However, similar gene therapies in neurons(nonproliferative cells that are non-susceptible to neoplasms) areproceeding rapidly, with active, FDA-approved clinical trials alreadyunderway involving viral gene delivery to human brains. This is likelyto facilitate the use of various embodiments of the present inventionfor a large variety of applications. The following is a non-exhaustivelist of a few examples of such applications and embodiments.

Addiction is associated with a variety of brain functions, includingreward and expectation. Additionally, the driving cause of addiction mayvary between individuals. According to one embodiment, addiction, forexample nicotine addiction, may be treated with optogeneticstabilization of small areas on the insula. Optionally, functional brainimaging—for example cued-state PET or fMRI—may be used to locate ahypermetabolic focus in order to determine a precise target spot for theintervention on the insula surface.

Optogenetic excitation of the nucleus accumbens and septum may providereward and pleasure to a patient without need for resorting to use ofsubstances, and hence may hold a key to addiction treatment. Conversely,optogenetic stabilization of the nucleus accumbens and septum may beused to decrease drug craving in the context of addiction. In analternative embodiment, optogenetic stabilization of hypermetabolicactivity observed at the genu of the anterior cingulate (BA32) can beused to decrease drug craving. Optogenetic stabilization of cells withinthe arcuate nucleus of the medial hypothalamus which contain peptideproducts of pro-opiomelanocortin (POMC) andcocaine-and-amphetamine-regulating transcript (CART) can also be used todecrease drug addiction behavior. For further information in thisregard, reference may be made to: Naqvi N H, Rudrauf D, Damasio H,Bechara A. “Damage to the insula disrupts addiction to cigarettesmoking.” Science. 2007 Jan. 26; 315(5811):531-534, which is fullyincorporated herein by reference.

Optogenetic stimulation of neuroendocrine neurons of the hypothalamicperiventricular nucleus that secrete somatostatin can be used to inhibitsecretion of growth hormone from the anterior pituitary, for example inacromegaly. Optogenetic stabilization of neuroendocrine neurons thatsecrete somatostatin or growth hormone can be used to increase growthand physical development. Among the changes that accompany “normal”aging, is a sharp decline in serum growth hormone levels after the4^(th) and 5^(th) decades. Consequently, physical deteriorationassociated with aging may be lessened through optogenetic stabilizationof the periventricular nucleus.

Optogenetic stabilization of the ventromedial nucleus of thehypothalamus, particularly the pro-opiomelanocortin (POMC) andcocaine-and-amphetamine-regulating transcript (CART) of the arcuatenucleus, can be used to increase appetite, and thereby treat anorexianervosa. Alternatively, optogenetic stimulation of the lateral nuclei ofthe hypothalamus can be used to increase appetite and eating behaviors.

Optogenetic excitation in the cholinergic cells of affected areasincluding the temporal lobe, the NBM (Nucleus basalis of Meynert) andthe posterior cingulate gyrus (BA 31) provides stimulation, and henceneurotrophic drive to deteriorating areas. Because the affected areasare widespread within the brain, an analogous treatment with implantedelectrodes may be less feasible than an opto-genetic approach.

Anxiety disorders are typically associated with increased activity inthe left temporal and frontal cortex and amygdala, which trends towardnormal as anxiety resolves. Accordingly, the affected left temporal andfrontal regions and amygdala may be treated with optogeneticstabilization, so as to dampen activity in these regions.

In normal physiology, photosensitive neural cells of the retina, whichdepolarize in response to the light that they receive, create a visualmap of the received light pattern. Optogenetic ion channels can be usedto mimic this process in many parts of the body, and the eyes are noexception. In the case of visual impairment or blindness due to damagedretina, a functionally new retina can be grown, which uses naturalambient light rather than flashing light patterns from an implanteddevice. The artificial retina grown may be placed in the location of theoriginal retina (where it can take advantage of the optic nerve servingas a conduit back to the visual cortex). Alternatively, the artificialretina may be placed in another location, such as the forehead, providedthat a conduit for the depolarization signals are transmitted tocortical tissue capable of deciphering the encoded information from theoptogenetic sensor matrix. Cortical blindness could also be treated bysimulating visual pathways downstream of the visual cortex. Thestimulation would be based on visual data produced up stream of thevisual cortex or by an artificial light sensor.

Treatment of tachycardia may be accomplished with optogeneticstimulation to parasympathetic nervous system fibers including CN X orVagus Nerve. This causes a decrease in the SA node rate, therebydecreasing the heart rate and force of contraction. Similarly,optogenetic stabilization of sympathetic nervous system fibers withinspinal nerves T1 through T4, serves to slow the heart. For the treatmentof pathological bradycardia, optogenetic stabilization of the Vagusnerve, or optogenetic stimulation of sympathetic fibers in T1 through T4will serve to increase heart rate. Cardiac disrhythmias resulting fromaberrant electrical foci that outpace the sinoatrial node may besuppressed by treating the aberrant electrical focus with moderateoptogenetic stabilization. This decreases the intrinsic rate of firingwithin the treated tissue, and permits the sinoatrial node to regain itsrole in pacing the heart's electrical system. In a similar way, any typeof cardiac arrhythmia could be treated. Degeneration of cardiac tissuethat occurs in cardiomyopathy or congestive heart failure could also betreated using this invention; the remaining tissue could be excitedusing various embodiments of the invention.

Optogenetic excitation stimulation of brain regions including thefrontal lobe, parietal lobes and hippocampi, may increase processingspeed, improve memory, and stimulate growth and interconnection ofneurons, including spurring development of neural progenitor cells. Asan example, one such application of the present invention is directed tooptogenetic excitation stimulation of targeted neurons in the thalamusfor the purpose of bringing a patient out of a near-vegetative(barely-conscious) state. Growth of light-gated ion channels or pumps inthe membrane of targeted thalamus neurons is effected. These modifiedneurons are then stimulated, e.g., via optics which may also gain accessby the same passageway, by directing a flash of light thereupon so as tomodulate the function of the targeted neurons and/or surrounding cells.For further information regarding appropriate modulation techniques (viaelectrode-based treatment) or further information regarding theassociated brain regions for such patients, reference may be made to:Schiff N D, Giacino J T, Kalmar K, Victor J D, Baker K, Gerber M, FritzB, Eisenberg B, O'Connor J O, Kobylarz E J, Farris S, Machado A, McCaggC, Plum F, Fins J J, Rezai A R. Behavioral improvements with thalamicstimulation after severe traumatic brain injury. Nature. Vol 448. Aug.2, 2007 pp 600-604.

In an alternative embodiment, optogenetic excitation may be used totreat weakened cardiac muscle in conditions such as congestive heartfailure. Electrical assistance to failing heart muscle of CHF isgenerally not practical, due to the thin-stretched, fragile state of thecardiac wall, and the difficulty in providing an evenly distributedelectrical coupling between an electrodes and muscle. For this reason,preferred methods to date for increasing cardiac contractility haveinvolved either pharmacological methods such as Beta agonists, andmechanical approaches such as ventricular assist devices. In thisembodiment of the present invention, optogenetic excitation is deliveredto weakened heart muscle via light emitting elements on the innersurface of a jacket surround the heart or otherwise against the affectedheart wall. Light may be diffused by means well known in the art, tosmoothly cover large areas of muscle, prompting contraction with eachlight pulse.

Optogenetic stabilization in the subgenual portion of the cingulategyrus (Cg25), yellow light may be applied with an implanted device. Thegoal would be to treat depression by suppressing target activity inmanner analogous to what is taught by Mayberg H S et al., Deep BrainStimulation for Treatment-Resistant Depression. Neuron, Vol. 45,651-660, Mar. 3, 2005, 651-660, which is fully incorporated herein byreference. In an alternative embodiment, an optogenetic excitationstimulation method is to increase activity in that region in a manneranalogous to what is taught by Schlaepfer et al., Deep Brain stimulationto Reward Circuitry Alleviates Anhedonia in Refractory Major Depression,Neuropsychopharmacology 2007 1-10, which is fully incorporated herein byreference. In yet another embodiment the left dorsolateral prefrontalcortex (LDPFC) is targeted with an optogenetic excitation stimulationmethod. Pacing the LDLPFC at 5-20 Hz serves to increase the basalmetabolic level of this structure which, via connecting circuitry,serves to decrease activity in Cg 25, improving depression in theprocess. Suppression of the right dorsolateral prefrontal cortex(RDLPFC) is also an effective depression treatment strategy. This may beaccomplished by optogenetic stabilization on the RDLPFC, or suppressionmay also be accomplished by using optogenetic excitation stimulation,and pulsing at a slow rate—1 Hz or less, improving depression in theprocess. Vagus nerve stimulation (VNS) may be improved using anoptogenetic approach. Use of optogenetic excitation may be used in orderto stimulate only the vagus afferents to the brain, such as the nodoseganglion and the jugular ganglion. Efferents from the brain would notreceive stimulation by this approach, thus eliminating some of theside-effects of VNS including discomfort in the throat, a cough,difficulty swallowing and a hoarse voice. In an alternative embodiment,the hippocampus may be optogenetically excited, leading to increaseddendritic and axonal sprouting, and overall growth of the hippocampus.Other brain regions implicated in depression that could be treated usingthis invention include the amygdala, accumbens, orbitofrontal andorbitomedial cortex, hippocampus, olfactory cortex, and dopaminergic,serotonergic, and noradrenergic projections. Optogenetic approachescould be used to control spread of activity through structures like thehippocampus to control depressive symptoms.

So long as there are viable alpha and beta cell populations in thepancreatic islets of Langerhans, the islets can be targeted for thetreatment of diabetes. For example, when serum glucose is high (asdetermined manually or by closed loop glucose detection system),optogenetic excitation may be used to cause insulin release from thebeta cells of the islets of Langerhans in the pancreas, whileoptogenetic stabilization is used to prevent glucagon release from thealpha cells of the islets of Langerhans in the pancreas. Conversely,when blood sugars are too low (as determined manually or by closed loopglucose detection system), optogenetic stabilization may be used to stopbeta cell secretion of insulin, and optogenetic excitation may be usedto increase alpha-cell secretion of glucagon.

For treatment of epilepsy, quenching or blocking epileptogenic activityis amenable to optogenetic approaches. Most epilepsy patients have astereotyped pattern of activity spread resulting from an epileptogenicfocus Optogenetic stabilization could be used to suppress the abnormalactivity before it spreads or truncated it early in its course.Alternatively, activation of excitatory tissue via optogeneticexcitation stimulation could be delivered in a series of deliberatelyasynchronous patterns to disrupt the emerging seizure activity. Anotheralternative involves the activation of optogenetic excitationstimulation in GABAergic neurons to provide a similar result. Thalamicrelays may be targeted with optogenetic stabilization triggered when anabnormal EEG pattern is detected.

Another embodiment involves the treatment of gastrointestinal disorders.The digestive system has its own, semi-autonomous nervous systemcontaining sensory neurons, motor neurons and interneurons. Theseneurons control movement of the GI tract, as well as trigger specificcells in the gut to release acid, digestive enzymes, and hormonesincluding gastrin, cholecystokinin and secretin. Syndromes that includeinadequate secretion of any of these cellular products may be treatedwith optogenetic stimulation of the producing cell types, or neuronsthat prompt their activity. Conversely, optogenetic stabilization may beused to treat syndromes in which excessive endocrine and exocrineproducts are being created. Disorders of lowered intestinal motility,ranging from constipation (particularly in patients with spinal cordinjury) to megacolan may be treated with optogenetic excitation of motorneurons in the intestines. Disorders of intestinal hypermotility,including some forms of irritable bowel syndrome may be treated withoptogenetic stabilization of neurons that control motility. Neurogenticgastric outlet obstructions may be treated with optogeneticstabilization of neurons and musculature in the pyloris. An alternativeapproach to hypomobility syndromes would be to provide optogeneticexcitation to stretch-sensitive neurons in the walls of the gut,increasing the signal that the gut is full and in need of emptying.

In this same paradigm, an approach to hypermobility syndromes of the gutwould be to provide optogenetic stabilization to stretch receptorneurons in the lower GI, thus providing a “false cue” that the gut wasempty, and not in need of emptying. In the case of frank fecalincontinence, gaining improved control of the internal and externalsphincters may be preferred to slowing the motility of the entire tract.During periods of time during which a patient needs to hold feces in,optogenetic excitation of the internal anal sphincter will provide forretention. Providing optogenetic stimulation to the external sphinctermay be used to provide additional continence. When the patient isrequired to defecate, the internal anal sphincter, and then externalanal sphincter should be relaxed, either by pausing the optogeneticstimulation, or by adding optogenetic stabilization.

Conductive hearing loss may be treated by the use of optical cochlearimplants. Once the cochlea has been prepared for optogeneticstimulation, a cochlear implant that flashes light may be used.Sensorineural hearing loss may be treated through optical stimulation ofdownstream targets in the auditory pathway.

Another embodiment of the present invention is directed toward thetreatment of blood pressure disorders, such as hypertension.Baroreceptors and chemoreceptors in regions such as the aorta (aorticbodies and paraaortic bodies) and the carotid arteries (“caroticbodies”) participate the regulation of blood pressure and respiration bysending afferents via the vagus nerve (CN X), and other pathways to themedulla and pons, particularly the solitary tract and nucleus.Optogenetic excitation of the carotid bodies, aortic bodies, paraorticbodies, may be used to send a false message of “hypertension” to thesolitary nucleus and tract, causing it to report that blood pressureshould be decreased. Optogenetic excitation or stabilization directly toappropriate parts of the brainstem may also be used to lower bloodpressure. The opposite modality causes the optogenetic approach to serveas a pressor, raising blood pressure. A similar effect may also beachieved via optogenetic excitation of the Vagus nerve, or byoptogenetic stabilization of sympathetic fibers within spinal nervesT1-T4. In an alternative embodiment, hypertension may be treated withoptogenetic stabilization of the heart, resulting in decreased cardiacoutput and lowered blood pressure. According to another embodiment,optogentic stabilization of aldosterone-producing cells within theadrenal cortex may be used to decrease blood pressure. In yet anotheralternative embodiment, hypertension may be treated by optogeneticstabilization of vascular smooth muscle. Activating light may be passedtranscutaneously to the peripheral vascular bed.

Another example embodiment is directed toward the treatment ofhypothalamic-pituitary-adrenal axis disorders. In the treatment ofhypothyroidism, optogenetic excitation of parvocellular neuroendocrine,neurons in the paraventricular and anterior hypothalamic nuclei can beused to increase secretion of thyrotropin-releasing hormone (TRH). TRH,in turn, stimulates anterior pituitary to secrete TSH. Conversely,hyperthyroidism may be treated with optogenetic stabilization of theprovocellular neuroendocrine neurons. For the treatment of adrenalinsufficiency, or of Addison's disease, optogenetic excitation ofparvocellular neuroendocrine neurons in the supraoptic nucleus andparaventricular nuclei may be used to increase the secretion ofvasopressin, which, with the help of corticotropin-releasing hormone(CRH), stimulate anterior pituitary to secrete ACTH. Cushing syndrome,frequently caused by excessive ACTH secretion, may be treated withoptogenetic stabilization of the parvocellular neuroendocrine neurons ofsupraoptic nucleus via the same physiological chain of effects describedabove. Neuroendocrine neurons of the arcuate nucleus produce dopamine,which inhibits secretion of prolactin from the anterior pituitary.Hyperprolactinemia can therefore be treated via optogenetic excitation,while hypoprolactinemia can be treated with optogenetic stabilization ofthe neuroendocrine cells of the arcuate nucleus.

In the treatment of hyperautonomic states, for example anxietydisorders, optogenetic stabilization of the adrenal medulla may be usedto reduce norepinephrine output. Similarly, optogenetic stimulation ofthe adrenal medulla may be used in persons with need for adrenalinesurges, for example those with severe asthma, or disorders that manifestas chronic sleepiness.

Optogenetic stimulation of the adrenal cortex will cause release ofchemicals including cortisol, testosterone, and aldosterone. Unlike theadrenal medualla, the adrenal cortex receives its instructions fromneuroendocrine hormones secreted from the pituitary and hypothalamus,the lungs, and the kidneys. Regardless, the adrenal cortex is amenableto optogenetic stimulation. Optogenetic stimulation of thecortisol-producing cells of the adrenal cortex may be used to treatAddison's disease. Optogenetic stabilization of cortisol-producing cellsof the adrenal cortex may be used to treat Cushing's disease.Optogenetic stimulation of testosterone-producing cells may be used totreat disorders of sexual interest in women: Optogenetic stabilizationof those same cells may be used to decrease facial hair in women.Optogenetic stabilization of aldosterone-producing cells within theadrenal cortex may be used to decrease blood pressure. Optogeneticexcitation of aldosterone-producing cells within the adrenal cortex maybe used to increase blood pressure.

Ontogenetic excitation stimulation of specific affected brain regionsmay be used to increase processing speed, and stimulate growth andinterconnection of neurons, including spurring the maturation of neuralprogenitor cells. Such uses can be particularly useful for treatment ofmental retardation.

According to another embodiment of the present invention, various musclediseases and injuries can be treated. Palsies related to muscle damage,peripheral nerve damage and to dystrophic diseases can be treated withoptogenetic excitation to cause contraction, and optogeneticstabilization to cause relaxation. This latter relaxation viaoptogenetic stabilization approach can also be used to prevent musclewasting, maintain tone, and permit coordinated movement as opposingmuscle groups are contracted. Likewise, frank spasticity can be treatedvia optogenetic stabilization.

In areas as diverse as peripheral nerve truncation, stroke, traumaticbrain injury and spinal cord injury, there is a need to foster thegrowth of new neurons, and assist with their integration into afunctional network with other neurons and with their target tissue.Re-growth of new neuronal tracts may be encouraged via optogeneticexcitation, which serves to signal stem cells to sprout axons anddendrites, and to integrate themselves with the network. Use of anoptogenetic technique (as opposed to electrodes) prevents receipt ofsignals by intact tissue, and serves to ensure that new target tissuegrows by virtue of a communication set up with the developing neurons,and not with an artificial signal like current emanating from anelectrode.

Obesity can be treated with optogenetic excitation to the ventromedialnucleus of the hypothalamus, particularly the pro-opiomelanocortin(POMC) and cocaine-and-amphetamine-regulating transcript (CART) of thearcuate nucleus. In an alternative embodiment, obesity can be treatedvia optogenetic stabilization of the lateral nuclei of the hypothalamus.In another embodiment, optogenetic stimulation to leptin-producingcells, or to cells with leptin receptors within the hypothalamus may beused to decrease appetite and hence treat obesity.

Destructive lesions to the anterior capsule, and analogous DBS to thatregion are established means of treating severe, intractableobsessive-compulsive disorder 48 (OCD48). Such approaches may beemulated using optogenetic stabilization to the anterior limb of theinternal capsule, or to regions such as BA32 and Cg24 which showmetabolic decrease as OCD remits.

Chronic Pain can be treated using another embodiment of the presentinvention. Electrical stimulation methods include local peripheral nervestimulation, local cranial nerve stimulation and “subthreshold” motorcortex stimulation. Reasonable optogentic approaches include optogeneticstabilization at local painful sites. Attention to promoter selectionwould ensure that other sensory and motor fibers would be unaffected.Selective optogenetic excitation of interneurons at the primary motorcortex also may provide effective pain relief. Also, optogeneticstabilization at the sensory thalamus, (particularly medial thalamicnuclei), periventricular grey matter, and ventral raphe nuclei, may beused to produce pain relief. In an alternative embodiment, optogeneticstabilization of parvalbumin-expressing cells targeting as targetingstrategy, may be used to treat pain by decreasing Substance Pproduction. The release of endogenous opioids may be accomplished byusing optogenetic excitation to increase activity in the nucleusaccumbens. In an alternative embodiment, when POMC neurons of thearcuate nucleus of the medial hypothalamus are optogenetically excited,beta endorphin are increased, providing viable treatment approaches fordepression and for chronic pain.

Parkinson's Disease can be treated by expressing optogeneticstabilization in the glutamatergic neurons in either the subthalamicnucleus (STN) or the globus pallidus interna (GPi) using anexcitatory-specific promoter such as CaMKIIα, and apply optogeneticstabilization. Unlike electrical modulation in which all cell-types areaffected, only glutamatergic STN neurons would be suppressed.

Certain personality disorders, including the borderline and antisocialtypes, demonstrate focal deficits in brain disorders including“hypofrontality.” Direct or indirect optogenetic excitation of theseregions is anticipated to produce improvement of symptoms. Abnormalbursts of activity in the amygdala are also known to precipitate sudden,unprompted flights into rage: a symptom of borderline personalitydisorder, as well as other conditions, which can benefit fromoptogenetic stabilization of the amygdala. Optogenetic approaches couldimprove communication and synchronization between different parts of thebrain, including amygdala, striatum, and frontal cortex, which couldhelp in reducing impulsiveness and improving insight.

The amygdalocentric model of post-traumatic-stress disorder (PTSD)proposes that it is associated with hyperarousal of the amygdala andinsufficient top-down control by the medial prefrontal cortex and thehippocampus. Accordingly, PTSD may be treated with optoeeneticstabilization of the amyedale or hippocampus.

Schizophrenia is characterized by abnormalities including auditoryhallucinations. These might be treated by suppression of the auditorycortex using optogenetic stabilization. Hypofrontality associated withschizophrenia might be treated with optogenetic excitation in theaffected frontal regions. Optogenetic approaches could improvecommunication and synchronization between different parts of the brainwhich could help in reducing misattribution of self-generated stimuli asforeign.

Optogenetic stabilization of cells within the arcuate nucleus of themedial hypothalamus, which contain peptide products ofpro-opiomelanocortin (POMC) and cocaine-and-amphetamine-regulatingtranscript (CART) can be used to reduce compulsive sexual behavior.Optogentic excitation of cells within the arcuate nucleus of the medialhypothalamus which contain peptide products of pro-opiomelanocortin(POMC) and cocaine-and-amphetamine-regulating transcript (CART) may beused to increase sexual interest in the treatment of cases of disordersof sexual desire. In the treatment of disorders of hypoactive sexualdesire testosterone production by the testes and the adrenal glands canbe increased through optogenetic excitation of the pituitary gland.Optogentic excitation of the nucleus accumbens can be used for thetreatment of anorgasmia.

The suprachiasmatic nucleus secretes melatonin, which serves to regulatesleep/wake cycles. Optogenetic excitation to the suprachiasmic nucleuscan be used to increase melatonin production, inducing sleep, andthereby treating insomnia. Orexin (hypocretin) neurons strongly excitenumerous brain nuclei in order to promote wakefulness. Optogenteticexcitation of orexin-producing cell populations can be used to treatnarcolepsy, and chronic daytime sleepiness.

Optogenetic stimulation of the supraoptic nucleus may be used to inducesecretion of oxytocin, can be used to promote parturition duringchildbirth, and can be used to treat disorders of social attachment.

Like muscular palsies, the motor functions that have been de-afferentedby a spinal cord injury may be treated with optogenetic excitation tocause contraction, and optogenetic stabilization to cause relaxation.This latter relaxation via optogenetic stabilization approach may alsobe used to prevent muscle wasting, maintain tone, and permit coordinatedmovement as opposing muscle groups are contracted. Likewise, frankspasticity may be treated via optogenetic stabilization. Re-growth ofnew spinal neuronal tracts may be encouraged via optogenetic excitation,which serves to signal stem cells to sprout axons and dendrites, and tointegrate themselves with the network.

Stroke deficits include personality change, motor deficits, sensorydeficits, cognitive loss, and emotional instability. One strategy forthe treatment of stroke deficits is to provide optogenetic stimulationto brain and body structures that have been deafferented from excitatoryconnections. Similarly, optogenetic stabilization capabilities can beimparted on brain and body structures that have been deafferented frominhibitory connections.

Research indicates that the underlying pathobiology in Tourette'ssyndrome is a phasic dysfunction of dopamine transmission in corticaland subcortical regions, the thalamus, basal ganglia and frontal cortex.In order to provide therapy, affected areas are preferably firstidentified using techniques including functional brain imaging andmagnetoencephalography (MEG). Whether specifically identified or not,optogenetic stabilization of candidate tracts may be used to suppressmotor tics. Post-implantation empirical testing of device parametersreveals which sites of optogenetic stabilization, and which areunnecessary to continue.

In order to treat disorders of urinary or fecal incontinence optogeneticstabilization can be used to the sphincters, for example via optogeneticstabilization of the bladder detrussor smooth muscle or innervations ofthat muscle. When micturation is necessary, these optogenetic processesare turned off, or alternatively can be reversed, with optogeneticstabilization to the (external) urinary sphincter, and optogeneticexcitation of the bladder detrussor muscle or its innervations. When abladder has been deafferentated, for example, when the sacral dorsalroots are cut or destroyed by diseases of the dorsal roots such as tabesdorsalis in humans, all reflex contractions of the bladder areabolished, and the bladder becomes distended. Optogenetic excitation ofthe muscle directly can be used to restore tone to the detrussor,prevent kidney damage, and to assist with the micturition process. Asthe bladder becomes “decentralized” and hypersensitive to movement, andhence prone to incontinence, optogenetic stabilization to the bladdermuscle can be used to minimize this reactivity of the organ.

In order to selectively excite/inhibit a given population of neurons,for example those involved in the disease state of an illness, severalstrategies can be used to target the optogenetic proteins/molecules tospecific populations.

For various embodiments of the present invention, genetic targeting maybe used to express various optogenetic proteins or molecules. Suchtargeting involves the targeted expression of the optogeneticproteins/molecules via genetic control elements such as promoters (e.g.,Parvalbumin, Somatostatin, Cholecystokinin, GFAP), enhancers/silencers(e.g., Cytomaglovirus Immediate Early Enhancer), and othertranscriptional or translational regulatory elements (e.g. WoodchuckHepatitis Virus Post-transcriptional Regulatory Element). Permutationsof the promoter+enhancer+regulatory element combination can be used torestrict the expression of optogenetic probes to genetically-definedpopulations.

Various embodiments of the present invention may be implemented usingspatial/anatomical targeting. Such targeting takes advantage of the factthat projection patterns of neurons, virus or other reagents carryinggenetic information (DNA plasmids, fragments, etc), can be focallydelivered to an area where a given population of neurons project to. Thegenetic material will then be transported back to the bodies of theneurons to mediate expression of the optogenetic probes. Alternatively,if it is desired to label cells in a focal region, viruses or geneticmaterial may be focally delivered to the interested region to mediatelocalized expression.

Various gene delivery systems are useful in implementing one or moreembodiments of the present invention. One such delivery system isAdeno-Associated Virus (AAV). AAV can be used to deliver apromoter+optogenetic probe cassett to a specific region of interest. Thechoice of promoter will drive expression in a specific population ofneurons. For example, using the CaMKIIa promoter will drive excitatoryneuron specific expression of optogenetic probes. AAV will mediatelong-term expression of the optogenetic probe for at least 1 year ormore. To achieve more specificity, AAV may be pseudotyped with specificserotypes 1 to 8, with each having different trophism for different celltypes. For instance, serotype 2 and 5 is known to have goodneuron-specific trophism.

Another gene deliver mechanism is the use of a retrovirus. HIV or otherlentivirus-based retroviral vectors may be used to deliver apromoter+optogenetic probe cassette to a specific region of interest.Retroviruses may also be pseudotyped with the Rabies virus envelopeglycoprotein to achieve retrograde transport for labeling cells based ontheir axonal projection patterns. Retroviruses integrate into the hostcell's genome, therefore are capable of mediating permanent expressionof the optogenetic probes. Non-lentivirus based retroviral vectors canbe used to selectively label dividing cells.

Gutless Adenovirus and Herpes Simplex Virus (HSV) are two DNA basedviruses that can be used to deliver promoter+optogenetic probe cassetteinto specific regions of the brain as well. HSV and Adenovirus have muchlarger packaging capacities and therefore can accommodate much largerpromoter elements and can also be used to deliver multiple optogeneticprobes or other therapeutic genes along with optogenetic probes.

Focal Electroporation can also be used to transiently transfect neurons.DNA plasmids or fragments can be focally delivered into a specificregion of the brain. By applying mild electrical current, surroundinglocal cells will receive the DNA material and expression of theoptogenetic probes.

In another instance, lipofection can be used by mixing genetic materialwith lipid reagents and then subsequently injected into the brain tomediate transfect of the local cells.

Various embodiments involve the use of various control elements. Inaddition to genetic control elements, other control elements(particularly promoters and enhancers whose activities are sensitive tochemical, magnetic stimulation, or infrared radiation) can be used tomediate temporally-controlled expression of the optogenetic probes. Forexample, a promoter whose transcriptional activity is subject toinfrared radiation allows one to use focused radiation to fine tune theexpression of optogenetic probes in a focal region at only the desiredtime.

According to one embodiment of the present invention, the invention maybe used in animal models of DBS, for example in Parkinsonian rats, toidentify the target cell types responsible for therapeutic effects (anarea of intense debate and immense clinical importance). This knowledgealone may lead to the development of improved pharmacological andsurgical strategies for treating human disease.

According to another embodiment of the present invention,genetically-defined cell types may be linked with complex systems-levelbehaviors, and may allow the elucidation of the precise contribution ofdifferent cell types in many different brain regions to high-levelorganismal functioning.

Other aspects and embodiments are directed to systems, methods, kits,compositions of matter and molecules for ion pumps or for controllinginhibitory currents in a cell (e.g., in in vivo and in vitroenvironments). As described throughout this disclosure, including theclaims, such systems, methods, kits, compositions of matter are realizedin manners consistent herewith. For example, in one embodiment, thepresent invention is directed to an assembly or kit of parts, having aproduct containing an NpHR-based molecular variant and anotheropsin-based molecule (ChR2-based and or NpHR-based) as a combinedpreparation for use in the treatment of disease of a neurological or CNSdisorder (as a category of disorder types or a specific disorder asexemplified herein), wherein at least the NpHR-based molecular variantis useful for expressing a light-activated NpHR-based molecule thatresponds to light by producing an inhibitory current to dissuadedepolarization of a cell, and wherein a high expression of the moleculemanifests a toxicity level that is less than about 75%.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the invention.Based on the above discussion and illustrations, those skilled in theart will readily recognize that various modifications and changes may bemade to the present invention without strictly following the exemplaryembodiments and applications illustrated and described herein. Forinstance, such changes may include additional NPHR-based sequences otherthan those listed in the immediately following Appendix. Suchmodifications and changes do not depart from the true spirit and scopeof the present invention, which is set forth in the following appendedclaims.

What is claimed is:
 1. A method for optically controlling a neuron, the method comprising exposing the neuron to light, wherein the neuron is genetically modified with an isolated nucleic acid comprising a mammalian codon optimized nucleotide sequence that encodes a variant opsin polypeptide derived from Natromonas pharaonis (NpHR), wherein the variant NpHR polypeptide comprises a heterologous endoplasmic reticulum (ER) export signal comprising the amino acid sequence set forth in SEQ ID NO:16, wherein the variant NpHR polypeptide inhibits depolarization of a neuron in response to the light, and wherein the variant NpHR polypeptide exhibits reduced toxicity in the neuron compared to toxicity induced by wild-type NpHR.
 2. The method of claim 1, wherein the variant NpHR polypeptide comprises a heterologous signal peptide.
 3. The method of claim 2, wherein the heterologous signal peptide is a nicotinic acetylcholine receptor signal peptide.
 4. The method of claim 3, wherein the heterologous signal peptide comprises the amino acid sequence set forth in SEQ ID NO:13.
 5. The method of claim 1, wherein the nucleic acid is present in a recombinant expression vector.
 6. The method of claim 5, wherein the nucleotide sequence is operably linked to a neuron-specific promoter.
 7. The method of claim 6, wherein the neuron-specific promoter is a CaMKIIα promoter.
 8. The method of claim 6, wherein the neuron-specific promoter is a cholinergic neuron-specific promoter.
 9. The method of claim 5, wherein the expression vector is a viral expression vector.
 10. The method of claim 9, wherein the viral expression vector is an adeno-associated viral vector or a lentivirus vector.
 11. The method of claim 10, wherein the viral expression vector is a lentivirus vector.
 12. The method of claim 1, wherein the variant NpHR polypeptide is encoded by nucleotides 1-873 of SEQ ID NO:1.
 13. The method of claim 1, wherein the neuron is in a brain region selected from the group consisting of amygdala, accumbens, cortex, hippocampus, dopaminergic projection, serotonergic projection, and noradrenergic projection.
 14. The method of claim 1, wherein the light has a wavelength of from 573 nm to 613 nm.
 15. The method of claim 1, wherein the neuron is further genetically modified to express a channelrhodopsin-2 light-activated polypeptide. 